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Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.
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Anticorpos , Reprodutibilidade dos Testes , Imuno-Histoquímica , Citometria de Fluxo , Especificidade de AnticorposRESUMO
Tendons are classified as dense fibrous connective tissue. This fibrous composition poses challenges in protein extraction, particularly hindering the application of Western blotting techniques. Because of these challenges, it becomes necessary to implement additional steps and specific solutions to attain success in this methodology with the tissue in question. The objective of this article is to provide a detailed protocol, elucidating each step, and making it easily replicable for researchers. The study focused on the Achilles tendons of Sprague-Dawley rats, emphasizing the need for a tailored approach in working with this tissue. By addressing the nuances of protein extraction from the dense and fibrous tendons, our protocol aims to facilitate the reproducibility of Western blotting experiments, contributing to a better understanding of this tissue.
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PURPOSE: The contribution of androgen receptors (AR) on bladder cancer has been demonstrated in pre-clinical studies, however in clinical studies, only the canonical AR (AR-FL) protein was measured by immunohistochemistry and conflicting results were obtained. To get better insight into the alterations of AR signalling, we used western blotting (WB) method and simultaneously measured both mRNA and protein levels of AR-FL and AR-V7. METHODS: 23 naive non-muscle invasive bladder cancer patients and 12 healthy individuals were included. AR-FL protein, AR-FL mRNA, AR-V7 protein and AR-V7 mRNA levels were quantitatively measured by WB and qRT-PCR. RESULTS: While AR-FL protein and AR-V7 mRNA were significantly higher in bladder cancer, AR-FL mRNA and AR-V7 protein were lower. AR-V7 mRNA level was higher in patients with tumour size over 3 cm and AR-FL protein was higher in single tumours (p < 0,005). The small sampling size and the inclusion of only male participants were the main limitations. CONCLUSIONS: The increase of AR-FL protein in bladder cancer supports the contribution of the AR pathway in bladder cancer. The presence of high AR-FL protein despite low mRNA levels may be due to a disruption in post-transcriptional regulatory mechanisms. AR-V7 was demonstrated for the first time in bladder tissue and found significantly different in bladder cancer tissues. Our study reached new and valuable findings and will shed light on the studies that aim to clarify the role of the AR pathway in bladder cancer.
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Receptores Androgênicos , Neoplasias da Bexiga Urinária , Humanos , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Receptores Androgênicos/genética , Masculino , Pessoa de Meia-Idade , Idoso , Feminino , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Isoformas de Proteínas/genética , Idoso de 80 Anos ou maisRESUMO
A 68-year-old woman presented with difficulty finding words and writing characters. Neurological examination led to clinical diagnosis at onset of the logopenic variant of primary progressive aphasia accompanied with ideomotor apraxia, visuospatial agnosia on the right, and Gerstmann syndrome. Bradykinesia and rigidity on the right with shuffling gait developed after one year. Treatment with L-dopa had no effect. The patient was diagnosed with corticobasal syndrome (CBS). Brain magnetic resonance imaging revealed diffuse cortical atrophy dominantly on the left, especially in the temporal, parietal, and occipital lobes. Positron emission tomography did not reveal any significant accumulation of amyloid ß or tau protein. She died five years later. Neuropathological examination revealed diffuse cortical atrophy with severe neuronal loss and fibrous gliosis in the cortex. Neuronal cytoplasmic inclusions, short dystrophic neurites, and, most notably, neuronal intranuclear inclusions, all immunoreactive for phosphorylated TDP-43, were observed. Western blotting revealed a full length and fragments of phosphorylated TDP-43 at 45 and 23 kDa, respectively, confirming the pathological diagnosis of type A FTLD-TDP. Whole exome sequencing revealed a pathogenic mutation in GRN (c.87dupC). FTLD-TDP should be included in the differential diagnosis of CBS.
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To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.
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Periploca , Ratos , Humanos , Animais , Espécies Reativas de Oxigênio/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Fluoretos/toxicidade , Fluoretos/metabolismo , Fígado/metabolismo , Apoptose , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Superóxido Dismutase/metabolismo , Estresse OxidativoRESUMO
The utility of serum glial fibrillary acidic protein (GFAP) in acute ischemic stroke (AIS) has been extensively studied in recent years. Here, we aimed to assess its potential role as a cargo protein of extracellular vesicles (EVs) secreted by astrocytes (ADEVs) in response to brain ischemia. Plasma samples from eighteen AIS patients at 24 h (D1), 7 days (D7), and one month (M1) post-symptoms onset, and nine age, sex, and cardiovascular risk factor-matched healthy controls were obtained to isolate EVs using the Exoquick ULTRA EV kit. Subsets of presumed ADEVs were identified further by the expression of the glutamate aspartate transporter (GLAST) as a specific marker of astrocytes with the Basic Exo-Flow Capture kit. Western blotting has tested the presence of GFAP in ADEV cargo. Post-stroke ADEV GFAP levels were elevated at D1 and D7 but not M1 compared to controls (p = 0.007, p = 0.019, and p = 0.344, respectively). Significant differences were highlighted in ADEV GFAP content at the three time points studied (n = 12, p = 0.027) and between D1 and M1 (z = 2.65, p = 0.023). A positive correlation was observed between the modified Rankin Scale (mRS) at D7 and ADEV GFAP at D1 (r = 0.58, p = 0.010) and D7 (r = 0.57, p = 0.013), respectively. ADEV GFAP may dynamically reflect changes during the first month post-ischemia. Profiling ADEVs from peripheral blood could provide a new way to assess the central nervous system pathology.
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Astrócitos , Vesículas Extracelulares , Proteína Glial Fibrilar Ácida , AVC Isquêmico , Humanos , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/sangue , Vesículas Extracelulares/metabolismo , Masculino , Feminino , AVC Isquêmico/metabolismo , AVC Isquêmico/sangue , Astrócitos/metabolismo , Projetos Piloto , Idoso , Pessoa de Meia-Idade , Biomarcadores/sangue , Idoso de 80 Anos ou mais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/sangue , Estudos de Casos e ControlesRESUMO
In the pathological process of Alzheimer's disease, neuronal cell death is closely related to the accumulation of reactive oxygen species. Our previous studies have found that oxidative stress can activate microtubule affinity-regulating kinases, resulting in elevated phosphorylation levels of tau protein specifically at the Ser262 residue in N1E-115 cells that have been subjected to exposure to hydrogen peroxide. This process may be one of the pathogenic mechanisms of Alzheimer's disease. Vitamin E is a fat-soluble, naturally occurring antioxidant that plays a crucial role in biological systems. This study aimed to examine the probable processes that contribute to the inhibiting effect on the abnormal phosphorylation of tau protein and the neuroprotective activity of a particular type of vitamin E, α-tocotrienol. The experimental analysis revealed that α-tocotrienol showed significant neuroprotective effects in the N1E-115 cell line. Our data further suggest that one of the mechanisms underlying the neuroprotective effects of α-tocotrienol may be through the inhibition of microtubule affinity-regulated kinase activation, which significantly reduces the oxidative stress-induced aberrant elevation of p-Tau (Ser262) levels. These results indicate that α-tocotrienol may represent an intriguing strategy for treating or preventing Alzheimer's disease.
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Neurônios , Fármacos Neuroprotetores , Estresse Oxidativo , Vitamina E , Proteínas tau , Proteínas tau/metabolismo , Fosforilação/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Vitamina E/farmacologia , Vitamina E/análogos & derivados , Fármacos Neuroprotetores/farmacologia , Animais , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Linhagem Celular Tumoral , TocotrienóisRESUMO
The expression of glutamate decarboxylase GAD65/67, an enzyme of GABA synthesis, and vesicular glutamate transporter 2 (VGLUT2) in the arcuate, dorsomedial, and ventromedial nuclei of the hypothalamus of young (3 months), adult (12 months), and old male rats (24 months) was studied by Western blotting. In old rats, an increase in the expression of GAD65/67 in the arcuate and dorsomedial, VGLUT2 in the arcuate, dorsomedial, and ventromedial nuclei was observed. Thus, an increase in opposite processes of inhibition and excitation is observed in the hypothalamic nuclei during aging.
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Hipotálamo , Neurônios , Animais , Masculino , Ratos , Envelhecimento/genética , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismoRESUMO
The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot.
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Anticorpos , Antígenos , Especificidade de Anticorpos , Western Blotting , CitocinasRESUMO
The exquisite balance between cellular prosurvival and death pathways is extremely necessary for homeostasis. Different forms of programmed cell death have been widely studied and reported such as apoptosis, necroptosis, pyroptosis, and autophagy. Autophagy is a catabolic process important for normal cellular functioning. The main aim of this machinery is to degrade the misfolded or damaged proteins, unuseful organelles, and pathogens, which invade the cells, thereby maintaining cellular homeostasis and assuring the regular renewal of cell components. This prosurvival function of autophagy highlights its importance in many human diseases, as the disturbance of this tightly organized process ultimately causes detrimental effects. Interestingly, neurons are particularly susceptible to damage upon the presence of any alteration in the basal level of the autophagic activity; this could be due to their high metabolic demand, post-mitotic nature, and the contribution of autophagy in the different fundamental functions of neurons. Herein, we have reported the role of autophagy in different CNS disorders such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and epilepsy, besides the pharmacological agents targeting autophagy. Due to the significant contribution of autophagy in the pathogenesis of many diseases, it is crucial to develop effective methods to detect this dynamic process. In this chapter, we have summarized the most frequently employed techniques in studying and detecting autophagy including electron microscopy, fluorescence microscopy, Western blotting, intracellular protein degradation, and sequestration assay.
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BACKGROUND: Limb Girdle Muscular Dystrophy R1 (LGMDR1) is an autosomal recessive neuromuscular disease caused by mutations in the calpain-3 (CAPN3) gene. As clinical and pathological features may overlap with other types of LGMD, therefore definite molecular diagnosis is required to understand the progression of this debilitating disease. This study aims to identify novel variants of CAPN3 gene in LGMDR1 patients. RESULTS: Thirty-four patients with clinical and histopathological features suggestive of LGMD were studied. The muscle biopsy samples were evaluated using Enzyme histochemistry, Immunohistochemistry, followed by Western Blotting and Sanger sequencing. Out of 34 LGMD cases, 13 patients were diagnosed as LGMDR1 by immunoblot analysis, demonstrating reduced or absent calpain-3 protein as compared to controls. Variants of CAPN3 gene were also found and pathogenicity was predicted using in-silico prediction tools. The CAPN3 gene variants found in this study, included, two missense variants [CAPN3: c.1189T > C, CAPN3: c.2338G > C], one insertion-deletion [c.1688delinsTC], one splice site variant [c.2051-1G > T], and one nonsense variant [c.1939G > T; p.Glu647Ter]. CONCLUSIONS: We confirmed 6 patients as LGMDR1 (with CAPN3 variants) from our cohort and calpain-3 protein expression was significantly reduced by immunoblot analysis as compared to control. Besides the previously known variants, our study found two novel variants in CAPN3 gene by Sanger sequencing-based approach indicating that genetic variants in LGMDR1 patients may help to understand the etiology of the disease and future prognostication.
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Calpaína , Distrofia Muscular do Cíngulo dos Membros , Humanos , Calpaína/genética , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Mutação/genética , Mutação de Sentido Incorreto , ProteômicaRESUMO
The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation. Because it is essential to understand the disease of giant panda for conservation, histopathological analyses of tissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors, which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognized N-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) in flow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 × 10-8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells.
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Anticorpos Monoclonais , Ursidae , Cricetinae , Camundongos , Animais , Humanos , Cricetulus , Células CHO , Células Endoteliais/metabolismo , Glicoproteínas de Membrana , Especificidade de Anticorpos , Fatores de TranscriçãoRESUMO
Extracranial waste transport from the brain interstitial fluid to the deep cervical lymph node (dCLN) is not extensively understood. The present study aims to show the cranial nerves that have a role in the transport of brain lymphatics vessels (LVs), their localization, diameter, and number using podoplanin (PDPN) and CD31 immunohistochemistry (IHC) and Western blotting. Cranial nerve samples from 6 human cases (3 cadavers, and 3 autopsies) were evaluated for IHC and 3 autopsies for Western blotting. The IHC staining showed LVs along the optic, olfactory, oculomotor, trigeminal, facial, glossopharyngeal, accessory, and vagus nerves. However, no LVs present along the trochlear, abducens, vestibulocochlear, and hypoglossal nerves. The LVs were predominantly localized at the endoneurium of the cranial nerve that has motor components, and LVs in the cranial nerves that had sensory components were present in all 3 layers. The number of LVs accompanying the olfactory, optic, and trigeminal nerves was classified as numerous; oculomotor, glossopharyngeal, vagus, and accessory was moderate; and facial nerves was few. The largest diameter of LVs was in the epineurium and the smallest one was in the endoneurium. The majority of Western blotting results correlated with the IHC. The present findings suggest that specific cranial nerves with variable quantities provide a pathway for the transport of wastes from the brain to dCLN. Thus, the knowledge of the transport of brain lymphatics along cranial nerves may help understand the pathophysiology of various neurological diseases.
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Encéfalo , Nervos Cranianos , Humanos , Nervos Cranianos/fisiologia , Nervo Vago/fisiologia , Nervo Facial/fisiologia , Crânio , Nervo Trigêmeo/fisiologia , Nervo Hipoglosso , Nervo Glossofaríngeo/fisiologia , Nervo Oculomotor , Nervo AbducenteRESUMO
Programmed cell death-ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single-chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof-of-concept data for the effect of scFv using PDL1-expressing cells was lacking. In this study, we conducted two kinds of cell-based immunoassays, western blotting and enzyme-linked immunosorbent assay, using anti-PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell-based immunoassays to detect PDL1.
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Antígeno B7-H1 , Proteínas Recombinantes , Anticorpos de Cadeia Única , Humanos , Antígeno B7-H1/imunologia , Antígeno B7-H1/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Linhagem Celular Tumoral , Ensaio de Imunoadsorção EnzimáticaRESUMO
Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season.
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Criopreservação , Cabras , Melatonina , Estações do Ano , Preservação do Sêmen , Sêmen , Espermatozoides , Animais , Masculino , Melatonina/metabolismo , Melatonina/sangue , Cabras/fisiologia , Cabras/metabolismo , Sêmen/química , Sêmen/metabolismo , Espermatozoides/metabolismo , Espermatozoides/fisiologia , Preservação do Sêmen/veterinária , Criopreservação/veterinária , Receptor MT1 de Melatonina/metabolismo , Receptor MT1 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismo , Receptor MT2 de Melatonina/genética , Análise do Sêmen/veterinária , Receptores de Melatonina/metabolismoRESUMO
The genome of the dengue virus codes for a single polypeptide that yields three structural and seven non-structural (NS) proteins upon post-translational modifications. Among them, NS protein-3 (NS3) possesses protease activity, involved in the processing of the self-polypeptide and in the cleavage of host proteins. Identification and analysis of such host proteins as substrates of this protease facilitate the development of specific drugs. In vitro cleavage analysis has been applied, which requires homogeneously purified components. However, the expression and purification of both S3 and erythroid differentiation regulatory factor 1 (EDRF1) are difficult and unsuccessful on many occasions. EDRF1 was identified as an interacting protein of dengue virus protease (NS3). The amino acid sequence analysis indicates the presence of NS3 cleavage sites in this protein. As EDRF1 is a high-molecular-weight (~138 kDa) protein, it is difficult to express and purify the complete protein. In this protocol, we clone the domain of the EDRF1 protein (C-terminal end) containing the cleavage site and the NS3 into two different eukaryotic expression vectors containing different tags. These recombinant vectors are co-transfected into mammalian cells. The cell lysate is subjected to SDS-PAGE followed by western blotting with anti-tag antibodies. Data suggest the disappearance of the EDRF1 band in the lane co-transfected along with NS3 protease but present in the lane transfected with only EDRF1, suggesting EDRF1 as a novel substrate of NS3 protease. This protocol is useful in identifying the substrates of viral-encoded proteases using ex vivo conditions. Further, this protocol can be used to screen anti-protease molecules. Key features ⢠This protocol requires the cloning of protease and substrate into two different eukaryotic expression vectors with different tags. ⢠Involves the transfection and co-transfection of both the above recombinant vectors individually and together. ⢠Involves western blotting of the same PVDF membrane containing total proteins of the cell lysate with two different antibodies. ⢠Does not require purified proteins for the analysis of cleavage of any suspected substrate by the protease.
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Plumage color, a pivotal attribute delineating diverse Muscovy duck strains, assumes considerable significance within the field of Muscovy duck breeding research. This study extends the existing research by delving into the hereditary aspects of genes associated with plumage coloration in Muscovy ducks. The principal objective is to discern marker genes conducive to targeted breeding strategies based on plumage color, thereby furnishing indispensable technical foundations for the development of novel Muscovy duck varieties. Our investigation focused on scrutinizing the impact of MYOT and MB genes on the genetic expression of plumage color at both the RNA and protein levels in Muscovy ducks. The results elucidate that black Muscovy ducks manifest markedly elevated mRNA and protein expression levels of MYOT and MB genes in comparison to their white counterparts, indicating that both genes may play a constructive regulatory role in the context of plumage coloration in Muscovy ducks. The outcomes of this study delineate a discernible correlation between MYOT and MB genes and the plumage coloration in Muscovy ducks. Employing gene expression analysis, we successfully identified candidate genes that may be intricately linked to the determination of plumage color in these ducks.
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Tau protein in Alzheimer's disease (AD) and tauopathies becomes insoluble due to hyperphosphorylation, conformational alterations, and aggregation. To analyze insoluble tau and pathological tau species, this study employs a methodology that utilizes wild-type and transgenic tau mice (P310S Tau) tissue extraction using 1% Sarkosyl or N-Lauroylsarcosine sodium salt and the radio immunoprecipitation assay (RIPA) buffer. However, the commonly used methods to study the insoluble tau fraction using detergents like Sarkosyl and RIPA require a large amount of homogenate, which can pose challenges when dealing with small tissue samples. Additionally, the study employs immunohistochemistry to visualize and quantify the pathological tau species in the brain tissue of transgenic mice, aiming to identify and analyze pathological tau species such as hyperphosphorylated tau to further our understanding of tauopathies such as Alzheimer's disease.
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Doença de Alzheimer , Tauopatias , Camundongos , Animais , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Tauopatias/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças , Encéfalo/metabolismoRESUMO
The involvement of apoptosis in neurodegeneration can be detected by quantifying the apoptotic proteins in hippocampal lysate. Apoptosis can occur due to the overproduction of apoptotic proteins under the influence of external trigger or due to the overexpression of the apoptotic genes. Thus, the imbalance in the production of apoptotic proteins can be quantified using the Western blotting technique and the overexpression of apoptotic genes in hippocampal DNA can be quantified using the real-time quantification of mRNA expression of the apoptotic proteins. Here we provide the methodology of detecting the apoptosis-related proteins like Bax and Bcl-2 and their mRNA expression in hippocampal neurodegeneration. In this chapter, we have described the methodology for quantification of mRNA expression of these apoptosis-related proteins in the hippocampal lysate using the real-time quantitative polymerase chain reaction (qPCR) technique and the methodology of detection and characterization of respective protein expression in the hippocampal lysate using the Western blotting technique.
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Proteínas Reguladoras de Apoptose , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/genética , Hipocampo/metabolismo , RNA Mensageiro/metabolismoRESUMO
PURPOSE: Glioma-associated oncogene homolog-1 (GLI1) is amplified in human glioblastoma, and there is growing evidence suggesting its significant role in tumor development and metastasis. Our aim was to investigate the role of the GLI-1 gene in the progression of colorectal cancer (CRC) and its correlation with various clinicopathological features. Additionally, we examined the impact of the GLI-1 gene and other factors on the prognosis of CRC. METHODS: We analyzed a total of 98 confirmed CRC cases and adjacent normal tissue controls. Patients suspected of having colon cancer underwent a colonoscopy and targeted biopsy, while those with rectal cancer underwent CT scans and MRI. GLI1 expression was detected using real-time PCR assay, Western blotting, and immunohistochemistry. RESULTS: The GLI1 gene was observed to be overexpressed in tumor tissues at both the protein and mRNA levels (p < 0.05). In addition, GLI1 overexpression was significantly associated with various factors such as tumor invasion (T3/T4), presence of lymph nodes, lymph node metastasis (LNM), stage (III/IV), tumor site (colon), tumor size (≥ 3 cm), localization (nucleocytoplasmic), strong staining intensity and recurrence (p < 0.05). The results of survival analysis showed that the patients with overexpression of GLI1 had a significantly lower DFS rate which was 21 months compared to those with normal expression who had 31 months (p < 0.05). Moreover, individuals with early onset disease (15 months) were more likely to have cytoplasmic localization of the GLI1 gene as opposed to nucleo-cytoplasmic localization of GLI1 which presented late-onset disease( 23 months) (p < 0.05). Finally, Stage and PNI (p < 0.05) were found to independently affect outcomes of CRC according to Cox regression analysis. CONCLUSION: High expression of GLI-1 in CRC is associated with adverse pathology and poor prognosis for patients. The correlation between cytoplasmic localization of GLI-1 and reduced disease-free survival holds potential for guiding prognosis and treatment. Further research is needed to develop strategies targeting GLI-1 for improved outcomes.