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1.
Head Face Med ; 18(1): 16, 2022 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-35668488

RESUMO

BACKGROUND: 4-Hexylresorcinol (4HR) is a food additive and class I histone deacetylase inhibitor. In this study, we examined the effects of 4HR administration on the submandibular gland in a growing rat model. METHODS: Four-week-old rats were used in this study. The experimental group (nine males and eight females) received 12.8 mg/kg of 4HR weekly for 12 weeks. Ten rats (five males and five females) were used as controls. The submandibular glands of rats were collected 12 weeks after the first administration of 4HR. The weight of the glands was measured. Histological analysis, immunoprecipitation-high-performance liquid chromatography (IP-HPLC), and western blotting were performed. RESULTS: The weights of the rat submandibular glands were higher in the experimental groups than in the control group, especially in male rats (P < 0.05). The vascular endothelial growth factor (VEGF) and testosterone in the submandibular glands were more highly expressed in 4HR-treated male rats than in untreated rats, as detected by both western blotting and immunohistochemistry. The IP-HPLC results demonstrated that the expression levels of Ki67, epidermal growth factor, and testosterone in the submandibular glands were higher in 4HR-treated male rats than in untreated rats. CONCLUSIONS: This study demonstrated that the systemic administration of 4HR increased the weight of submandibular glands in male rats. In addition, the testosterone and VEGF expression levels in the submandibular glands increased owing to 4HR administration.


Assuntos
Hexilresorcinol , Animais , Western Blotting , Feminino , Hexilresorcinol/farmacologia , Humanos , Masculino , Ratos , Glândula Submandibular , Testosterona , Fator A de Crescimento do Endotélio Vascular
2.
J Vis Exp ; (183)2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35661678

RESUMO

The role of extracellular vesicles (EVs) in the context of bacterial infection has emerged as a new avenue for understanding microbial physiology. Specifically, Mycobacterium tuberculosis (Mtb) EVs play a role in the host-pathogen interaction and response to environmental stress. Mtb EVs are also highly antigenic and show potential as vaccine components. The most common method for purifying Mtb EVs is density gradient ultracentrifugation. This process has several limitations, including low throughput, low yield, reliance on expensive equipment, technical challenges, and it can negatively impact the resulting preparation. Size exclusion chromatography (SEC) is a gentler alternative method that combats many of the limitations of ultracentrifugation. This protocol demonstrates that SEC is effective for Mtb EV enrichment and produces high-quality Mtb EV preparations of increased yield in a rapid and scalable manner. Additionally, a comparison to density gradient ultracentrifugation by quantification and qualification procedures demonstrates the benefits of SEC. While the evaluation of EV quantity (nanoparticle tracking analysis), phenotype (transmission electron microscopy), and content (Western blotting) is tailored to Mtb EVs, the workflow provided can be applied to other mycobacteria.


Assuntos
Vesículas Extracelulares , Mycobacterium tuberculosis , Western Blotting , Cromatografia em Gel , Vesículas Extracelulares/química , Ultracentrifugação/métodos
3.
Anal Biochem ; 652: 114751, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35667451

RESUMO

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a standard method for protein analysis. However, the stacking gel employed for the standard SDS-PAGE is transparent and indistinct. Therefore, loading samples into stacking gel wells can be challenging. Accordingly, an acidic dye (tartrazine, brilliant blue FCF, or new coccine), which allowed easy visualization of the stacking gel wells and straightforward sample loading into these wells, was added to the SDS-PAGE stacking gel to resolve this issue. Moreover, the performance of these gels was comparable to that of non-colored standard gels. Thus, researchers worldwide could adopt this simple method for protein analysis.


Assuntos
Proteínas , Western Blotting , Eletroforese em Gel de Poliacrilamida , Géis , Proteínas/análise , Dodecilsulfato de Sódio
4.
Methods Enzymol ; 667: 59-77, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35525555

RESUMO

Protein kinases catalyze the transfer of a phosphate group thereby activating proteins and initiating signaling cascades. Their cousins, the pseudokinases, are enzymatically nonactive counterparts of protein kinases that can be considered zombie enzymes. Interestingly, pseudokinases, which constitute about 10% of the human kinome, have been implicated in many cancers, despite their sequences predicting a lack of catalytic activity. Owing to recent research, it has been demonstrated that dysregulation of many pseudokinases triggers changes in cell signaling, proliferation, and drug resistance. This review is aimed at describing methods that can be used for detection of Tribbles family of pseudokinases, specifically TRIB2. We describe intracellular staining by flow cytometry and Western blotting techniques for the detection of endogenous TRIB2 protein.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina , Peptídeos e Proteínas de Sinalização Intracelular , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citometria de Fluxo , Humanos
5.
Stem Cell Res Ther ; 13(1): 212, 2022 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-35619161

RESUMO

BACKGROUND: Endometrial carcinoma is the most common gynecological cancer in Europe. Musashi-1 is known to be a key regulator of endometrial cancer stem cells and a negative prognostic marker. In the present study, we aimed to understand growth and gene expression patterns in endometrial carcinoma after Musashi-1 knockdown in vitro and in vivo. Changes in therapeutic resistance were also assessed. METHODS: First, we performed analyses to understand Musashi-1 expression patterns using The Cancer Genome Atlas database. We then proceeded to assess effects of small interfering RNA-based Musashi-1 targeting in two endometrial carcinoma cell lines, Ishikawa and KLE. After quantifying baseline changes in cell metabolism, we used MTT tests to assess chemotherapy effects and colony formation assays to understand changes in radioresistance. For mechanistic study, we used quantitative polymerase chain reaction (qPCR) and western blotting of key Musashi-1 target genes and compared results to primary tissue database studies. Finally, xenograft experiments in a mouse model helped understand in vivo effects of Musashi-1 knockdown. RESULTS: Musashi-1 is aberrantly expressed in primary tumor tissues. In vitro, silencing of Musashi-1 resulted in a strong decline in cell proliferation and radioresistance, while chemoresistance remained unchanged. Loss of Musashi-1 led to downregulation of telomerase, DNA-dependent protein kinase, the Notch pathway and overexpression of cyclin-dependent kinase inhibitor p21, the latter of which we identified as a key mediator of Msi-1 knockdown-related anti-proliferative signaling. In vivo, the anti-proliferative effect was confirmed, with Msi-1 knockdown tumors being about 40% reduced in size. CONCLUSIONS: Musashi-1 knockdown resulted in a strong decrease in endometrial cancer proliferation and a loss of radioresistance, suggesting therapeutic potential.


Assuntos
Neoplasias do Endométrio , Animais , Biomarcadores , Western Blotting , Proliferação de Células/genética , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/radioterapia , Feminino , Humanos , Camundongos , Células-Tronco/metabolismo
6.
Pancreatology ; 22(5): 564-571, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35589511

RESUMO

OBJECTIVE: Non-alcoholic chronic pancreatitis (NACP) frequently develops in the setting of genetic susceptibility associated with alterations in genes that are highly expressed in the pancreas. However, the genetic basis of NACP remains unresolved in a significant number of patients warranting a search for further risk genes. DESIGN: We analyzed CUZD1, which encodes the CUB and zona pellucida-like domains 1 protein that is found in high levels in pancreatic acinar cells. We sequenced the coding region in 1163 European patients and 2018 European controls. In addition, we analyzed 297 patients and 1070 controls from Japan. We analyzed secretion of wild-type and mutant CUZD1 from transfected cells using Western blotting. RESULTS: In the European cohort, we detected 30 non-synonymous variants. Using different prediction tools (SIFT, CADD, PROVEAN, PredictSNP) or the combination of these tools, we found accumulation of predicted deleterious variants in patients (p-value range 0.002-0.013; OR range 3.1-5.2). No association was found in the Japanese cohort, in which 13 non-synonymous variants were detected. Functional studies revealed >50% reduced secretion of 7 variants, however, these variants were not significantly enriched in European CP patients. CONCLUSION: Our data indicate that CUZD1 might be a novel susceptibility gene for NACP. How these variants predispose to pancreatitis remains to be elucidated.


Assuntos
Pancreatite Crônica , Zona Pelúcida , Células Acinares/metabolismo , Western Blotting , Humanos , Proteínas de Membrana/genética , Pâncreas/metabolismo , Hormônios Pancreáticos , Pancreatite Crônica/genética , Zona Pelúcida/metabolismo
7.
Methods Mol Biol ; 2447: 127-137, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35583778

RESUMO

Metacaspases are cysteine proteases that are present in plants, protists, fungi, and bacteria. Previously, we found that physical damage, e.g., pinching with forceps or grinding on liquid nitrogen of plant tissues, activates Arabidopsis thaliana METACASPASE 4 (AtMCA4). AtMCA4 subsequently cleaves PROPEP1, the precursor pro-protein of the plant elicitor peptide 1 (Pep1). Here, we describe a protein extraction method to detect activation of AtMCA4 by Western blot with antibodies against endogenous AtMCA4 and a PROPEP1-YFP fusion protein. It is important to (1) keep plant tissues at all times on liquid nitrogen prior to protein extraction, and (2) denature the protein lysate as fast as possible, as metacaspase activation ensues quasi immediately because of tissue damage inherent to protein extraction. In theory, this method can serve to detect damage-induced alterations of any protein-of-interest in any organism for which antibodies or fusion proteins are available, and hence, will greatly aid the study of rapid damage-activated proteolysis in the future.


Assuntos
Arabidopsis , Arabidopsis/metabolismo , Western Blotting , Caspases/metabolismo , Nitrogênio/metabolismo , Plantas/metabolismo , Proteólise
8.
Exp Parasitol ; 238: 108268, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35513005

RESUMO

Neospora caninum is an obligate intracellular parasite related to abortion in cattle, goats and sheep. The life cycle of N. caninum is characterized by the time-coordinated secretion of proteins contained in micronemes, rhoptries and dense granules, allowing the active invasion and the adaptation of the parasite in the cell environment. Thus, the proteins of the secretome have the potential to be considered as targets for N. caninum control. Despite the importance of neosporosis in the livestock-related economy, no commercial treatment is available. Furthermore, the process of invasion, propagation and immune evasion are not completely elucidated. In this study, we initiated the characterization of NCLIV_011700 of N. caninum, a protein with low sequence identity to NcROP15 or TgROP15 (<15%). Our goal was the detection and molecular characterization of the NCLIV_011700, once homology (with low identity >20%) was observed within the Apicomplexa. The NCLIV_011700 sequence was aligned and compared to the closer apicomplexan homologues (ROP15 from N. caninum, T. gondii, Hammondia hammondi, Cystospores suis), including the predicted domains. In general, the NCLIV_011700 demonstrated low identity with ROP15 of apicomplexan (<20%) and had a ubiquitin domain. On the other side, the NCLIV_011700 homologues were composed of a non-cytoplasmic domain, suggesting different functions between NcROP15 (or homologues) and NCLIV_011700 during the parasite life cycle. Moreover, the NCLIV_011700 was amplified by PCR, ligated to a pET28a plasmid and expressed in Escherichia coli. The recombinant form of NCLIV_011700 was purified in a nickel-Sepharose resin and applied for polyclonal antibody production in mice. The antiserum against NCLIV_011700 (anti-rNCLIV_011700) was used to localize the native form of the protein using Western blot and confocal microscopy. Also, the NCLIV_011700 antiserum partially inhibited the parasite adhesion/invasion process, indicating an active role of the protein in the N. caninum cycle. Thus, the initial NCLIV_011700 characterization will contribute to enlarging the comprehension of N. caninum, aiming at the future development of tools to control the parasite infection/propagation.


Assuntos
Coccidiose , Neospora , Animais , Western Blotting , Bovinos , Coccidiose/parasitologia , Cabras , Camundongos , Neospora/genética , Reação em Cadeia da Polimerase , Proteínas , Ovinos
9.
PLoS One ; 17(5): e0268537, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35587943

RESUMO

When bovine lactoferrin (bLF) contacts human vaginal fluid (VF) it is subjected to proteolytic degradation. This report describes fragmentation patterns of bLF dosed vaginally in clinical trials or incubated ex vivo with VF. A consensus pattern of fragments was observed in samples from different women. The 80 kDa bLF molecule is initially cleaved between its homologous 40 kDa domains, the N-lobe and C-lobe, and then degraded into sub-fragments and mixtures of small peptides. We characterized this fragmentation process by polyacrylamide gel electrophoresis, western blotting, chromatographic separation, and mass spectral sequence analysis. Common to most VF fragmentation patterns were large amounts of an N-lobe 37 kDa fragment and a C-lobe 43 kDa fragment resulting from a single cleavage following tyrosine 324. Both fragments possessed full sets of iron-ligand amino acids and retained iron-binding ability. In some VF samples, alternative forms of large fragments were found, which like the 37+43 kDa pair, totaled 80 kDa. These included 58+22 kDa, 18+62 kDa, and 16+64 kDa forms. In general, the smaller component was from the N-lobe and the larger from the C-lobe. The 18+62 kDa pair was absent in some VF samples but highly abundant in others. This variability suggests multiple endopeptidases are involved, with the 18 kDa fragment's presence dependent upon the balance of enzymes. Further action of VF endopeptidases produced smaller peptide fragments, and we found evidence that exopeptidases trimmed their N- and C-termini. The 3.1 kDa antimicrobial peptide lactoferricin B was not detected. These studies were facilitated by a novel technique we developed: tricolor western blots, which enabled simultaneous visualization of N- and C-terminal epitopes.


Assuntos
Lactoferrina , Peptídeo Hidrolases , Western Blotting , Eletroforese em Gel de Poliacrilamida , Endopeptidases/metabolismo , Feminino , Humanos , Ferro/metabolismo , Lactoferrina/química , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 38(5): 452-459, 2022 May.
Artigo em Chinês | MEDLINE | ID: mdl-35603654

RESUMO

Objective To generate rabbit polyclonal antibody against mouse Tubby(Tub)-like protein 2 (TULP2) and detect the expression of TULP2 in mouse testis. Methods pET30a (+)-TULP2 and pET30(+)-TULP2-C recombinant plasmids were constructed by inserting TULP2 full-length gene fragment and TULP2-C gene fragment containing Tub domain into pET30a (+). pET30a (+)-TULP2 and pET30(+)-TULP2-C were transformed into E. coli BL21, and the prokaryotic protein expressions were induced with the supplementation of IPTG. The prokaryotic recombinant proteins were purified with His-Binding-resin, and denaturation was performed by adding urea with gradient concentration. Adult male New Zealand white rabbits were inoculated with recombinant TULP2 and TULP2-C proteins as immunogens to generate two kinds of TULP2 polyclonal antibodies. Titers of antibodies were detected by ELISA. The efficiency and specificity of antibodies were determined by Western blot and immunofluorescence (IF) staining. Results pET30a (+)-TULP2 and pET30a (+)-TULP2-C recombinant plasmids were constructed successfully, and the protein expressions of TULP2 and TULP2-C could be induced by adding IPTG. The titers of polyclonal antibodies were 1:1 000 000. Western blot and IF staining showed poor specificity of TULP2-C antibody. TULP2 antibody could specifically recognize the endogenous TULP2 protein in the testes of adult wild-type mice, and IF staining showed that TULP2 was expressed specifically in the round spermatids and elongating spermatids of mice. Conclusion A rabbit anti-mouse TULP2 polyclonal antibody is generated successfully using TULP2 full-length protein, which can be used for detecting TULP2 expression by Western blot and IF staining.


Assuntos
Anticorpos , Escherichia coli , Animais , Especificidade de Anticorpos , Western Blotting , Escherichia coli/genética , Isopropiltiogalactosídeo , Masculino , Camundongos , Coelhos
11.
Methods Mol Biol ; 2480: 17-48, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35616855

RESUMO

Nicotiana tabacum (the tobacco plant ) has numerous advantages for molecular farming, including rapid growth, large biomass and the possibility of both cross- and self-fertilization. In addition, genetic transformation and tissue culture protocols for regeneration of transgenic plants are well-established. Here, we describe the production of transgenic tobacco using Agrobacterium tumefaciens and the analysis of recombinant proteins, either in crude plant extracts or after purification, by enzyme-linked immunosorbent assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis with western blotting and surface plasmon resonance.


Assuntos
Agrobacterium tumefaciens , Tabaco , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Western Blotting , Plantas Geneticamente Modificadas , Proteínas Recombinantes/metabolismo , Tabaco/metabolismo
12.
Bull Exp Biol Med ; 172(6): 701-708, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35503584

RESUMO

It is known that the expression of the deubiquitinating enzyme BRCA1-BRCA2-containing complex subunit 3 (BRCC3) and cyclin-dependent protein kinase 5 (Cdk5) is increased in Parkinson's disease (both are involved in neuroinflammatory response). However, the regulatory mechanism of Cdk5 on the post-translational modification of BRCC3 remains unclear. Here we studied whether Cdk5 phosphorylates BRCC3. Phosphorylation of BRCC3 by Cdk5 was predicted by GPS 5.0 software. His-BRCC3 plasmid was constructed by cloning the BRCC3 gene into pGEX-6P-1 vector, and then His-BRCC3 fusion protein was induced with isopropyl ß-d-1-thiogalactopyranoside and purified using His-Tag affinity chromatography purification agarose. Phosphorylation of BRCC3 fusion protein by Cdk5 in vitro was detected by mass spectrometry and Western blotting. The results showed that multiple phosphorylation sites were predicted by GPS 5.0, and the His-BRCC3 fusion protein was successfully induced and purified. In vitro kinase assay, Western blotting, and mass spectrometry showed that Cdk5 can phosphorylate BRCC3. It has been demonstrated that protein kinase Cdk5 can phosphorylate the deubiquitinating enzyme BRCC3 in vitro, which provides new data for further study on the mechanism of neurodegeneration.


Assuntos
Quinase 5 Dependente de Ciclina , Enzimas Desubiquitinantes , Western Blotting , Quinase 5 Dependente de Ciclina/metabolismo , Enzimas Desubiquitinantes/genética , Enzimas Desubiquitinantes/metabolismo , Humanos , Doença de Parkinson/metabolismo , Fosforilação
14.
Am J Case Rep ; 23: e935717, 2022 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-35398874

RESUMO

BACKGROUND Many diagnostic guidelines have been established to support the diagnosis of Lyme disease, but a recent meta-analysis did not find that 2-tier tests were better than individual tests. Here, we present the case of a patient who was diagnosed by immunoblot only, a second-line test that is usually not performed if the first-line test is negative. CASE REPORT A 60-year-old Swiss woman, without relevant comorbidities, presented to our clinic with 1-week symptoms of migratory radiculitis in the L1, L2, and L5-S1 right dermatomes. Blood analysis and lumbar and brain MRI did not show any significant abnormalities. However, unexpected results were obtained after testing Lyme serologies. They were performed first with LIAISON® test (Diasorin, Italy) then with Borrelia VIRAstripe® immunoblot (Viramed, Germany) and a positive IgM result was only obtained with the latter. Consequently, doxycycline 100 mg 2×/day was initiated and the symptoms completely resolved after 6 weeks of treatment. Ever since, and more than 1 year after the initial presentation, the patient remains symptom-free. CONCLUSIONS As shown, it was possible to diagnose this patient and treat her successfully by testing all the available serologies. Furthermore, we were surprised to find out after a review of the literature that the IgM sensitivity in neuroborreliosis with the LIAISON® test is only 43.9-46% versus 90-100% with VIRAstripe®. Hence, clinicians need to understand the pitfalls of these tests before excluding Lyme disease.


Assuntos
Anticorpos Antibacterianos , Doença de Lyme , Western Blotting , Feminino , Humanos , Imunoglobulina G , Imunoglobulina M , Doença de Lyme/complicações , Doença de Lyme/diagnóstico , Doença de Lyme/tratamento farmacológico , Pessoa de Meia-Idade , Dor , Suíça
15.
Methods Mol Biol ; 2504: 101-112, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35467282

RESUMO

Exosomes are small extracellular vesicles secreted by cells and are known to play a key role in intercellular communication. Several studies have associated exosomes with various roles in tumorigenesis and explored their potential as a source of biomarkers for diagnosis and prognosis in cancer research. Exosomes can be isolated from several body fluids, including those that are noninvasively accessible, such as human saliva. This book chapter provides a step-by-step detailed description of techniques that are used for the isolation, quantification, and characterization of exosomes from saliva. These include ultracentrifugation for the isolation, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot (WB) for characterization of exosomes. The NTA approach explores the Brownian motion and light scattering of particles to predict size and concentration. TEM enables visualization of the exosomes which often present a cup-shaped morphology. Western blot is used to detect commonly expressed exosome-associated proteins. Finally, salivary exosomes isolated using these protocols can further be characterized for downstream analysis according to their cargo (proteins, DNA, RNA, miRNA) and utilized for cancer biomarker discovery.


Assuntos
Exossomos , MicroRNAs , Neoplasias , Biomarcadores Tumorais/metabolismo , Western Blotting , Exossomos/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Proteínas/metabolismo , Ultracentrifugação/métodos
16.
Pathol Res Pract ; 233: 153879, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35405623

RESUMO

BACKGROUND: Colorectal cancer (CRC) is a familiar malignancy accompanied by higher morbidity and mortality. The deubiquitination enzyme USP20 has been discovered to be one key factor in several cancers progression. SOX4 is a critical transcription factor to regulate the expression of various genes, and participates into the occurrence and progression of cancers. In this study, it was aimed to illustrate the role of USP20 and the regulatory relationship between USP20 and SOX4 in CRC. METHODS: The protein expressions of USP20, SOX4, E-cadherin, N-cadherin, Snail and slug were tested through western blot. The cell proliferation ability was verified through CCK-8 assay. The migration and invasion abilities were detected through Transwell assay. The mRNA expression of SOX4 was confirmed through RT-qPCR. The interaction between USP20 and SOX4 was notarized through Co-IP assay. RESULT: Our study demonstrated that USP20 displayed higher expression, and facilitated CRC progression through regulating cell proliferation, migration, invasion and EMT process markers. USP20 was found to modulate SOX4 protein expression. Next, it was verified that USP20 regulated SOX4 degradation through deubiquitination. Finally, through rescue assays, we revealed that USP20 mediated SOX4 expression to accelerate CRC progression. CONCLUSIONS: In this study, USP20 regulated the stability of EMT transcription factor SOX4 and aggravated colorectal cancer metastasis. This finding might highlight the function of USP20 in the treatment of CRC.


Assuntos
Neoplasias Colorretais , Fatores de Transcrição , Western Blotting , Proliferação de Células , Neoplasias Colorretais/genética , Regulação da Expressão Gênica , Humanos , Fatores de Transcrição SOXC/genética , Ubiquitina Tiolesterase
17.
Biochem Biophys Res Commun ; 607: 110-116, 2022 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-35367822

RESUMO

When performing western blots for protein detection using the classical Laemmli method, experimenters often encounter difficulties with the detection of transmembrane proteins involved in lipid or fatty acid metabolism. A crucial phase in sample preparation is heating the samples to 100 °C in a Laemmli sample buffer containing SDS before separation by polyacrylamide gel electrophoresis (PAGE). In the current study, the analysis of several proteins was performed following modifications of the heating step during sample preparation. Multiple samples of the human Jurkat cell line were prepared using commercial or homemade Laemmli sample buffer. Samples were subjected to incubation at different temperatures for varying periods of time prior to separation by SDS-PAGE, transfer onto PVDF membranes and detection with specific antibodies. In samples incubated at temperatures of 25 °C, 40 °C, 70 °C and 100 °C, detection of the transmembrane protein elongase of long chain fatty acids 5 (ELOVL5) significantly decreased with temperature to a near total absence of signal at 100 °C. Heating (100 °C) the samples even for 1 min resulted in significant loss of ELOVL5 band intensity that was associated with the appearance of higher molecular weight immunoreactive materials. Loss of ELOVL5 band intensity was also observed with heating of samples at 100 °C prepared from HepG2, HEK293, MCF-7 and SKRB cells. The robust induction of ELOVL5 in stimulated primary T cells was not detected when sample were heated. The detection of fatty acid-metabolizing enzymes stearoyl-CoA desaturase-1 and long-chain-fatty-acid-CoA ligases 3 and 4 showed bands with significantly less intensity after heating at 100 °C compared to samples prepared at room temperature. Heating samples at 100 °C did not affect the detection of transmembrane proteins ERBB2 and five-lipoxygenase activating protein, or the soluble 5-lipoxygenase protein. Overall, the number of transmembrane passes of a protein was not predictive of loss of band intensity after heating, however this study indicates that sample heating can drastically affect the ability to detect proteins following separation by SDS-PAGE. This has implications for any detection methods that follow SDS-PAGE.


Assuntos
Ácidos Graxos , Calefação , Western Blotting , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Proteínas
18.
STAR Protoc ; 3(2): 101274, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35403002

RESUMO

Membrane proteins (MPs) are essential in many cellular functions. To maintain proteostasis, MPs are downregulated via ubiquitination and degradation. Here, we describe an optimized protocol to analyze MP degradation using quantitative western blot and flow cytometry-based approaches. We use the degradation of Ypq1, a vacuole membrane lysine transporter, to demonstrate the protocol, which can be adapted for other organelle MPs and thus provide useful tools to study MP regulation in yeast and other model organisms. For complete details on the use and execution of this protocol, please refer to Arines et al. (2021) and Yang et al. (2020).


Assuntos
Proteínas de Membrana , Saccharomyces cerevisiae , Western Blotting , Citometria de Fluxo/métodos , Proteólise
19.
J Immunol Res ; 2022: 5473763, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35434142

RESUMO

In the past decade, the interest in helminth-derived extracellular vesicles (EVs) increased owing to their role in pathogen-host communication. However, the availability of EVs from these parasitic worms is often limited due to the restricted occurrence and culturing possibilities of these organisms. Schistosoma mansoni is one of several helminths that have been shown to release EVs affecting the immune response of their host. Further investigation of mechanisms underlying these EV-induced effects warrants separation of EVs from other components of the helminth excretory/secretory products. However, isolation of high-purity EVs often come to the expense of reduced EV yield. We therefore aimed to develop an optimized protocol for isolation of EVs from S. mansoni schistosomula and adult worms with respect to purity, concentration, and yield. We tested the use of small (1.7 ml) iodixanol density gradients and demonstrated that this enabled western blot-based analysis of the EV marker protein tetraspanin-2 (TSP-2) in gradient fractions without additional concentration steps. Moreover, the concentration and yield of EVs obtained with small iodixanol gradients were higher compared to medium-sized (4.3 ml) or conventional large-sized (12 ml) gradients. Additionally, we provide evidence that iodixanol is preferred over sucrose as medium for the small density gradients, because EVs in iodixanol gradients reached equilibrium much faster (2 hours) and iodixanol but not sucrose was suitable for purification of schistosomula EVs. Finally, we demonstrate that the small iodixanol gradients were able to separate adult worm EVs from non-EV contaminants such as the blood digestion product hemozoin. Our optimized small iodixanol density gradient allows to simultaneously separate and concentrate EVs while reducing handling time and EV loss and can be applied for EVs from helminths and other limited EV sources.


Assuntos
Vesículas Extracelulares , Schistosoma mansoni , Animais , Biomarcadores/metabolismo , Western Blotting , Proteínas
20.
Biotechniques ; 72(5): 207-218, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35383476

RESUMO

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(N-morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.


Assuntos
COVID-19 , SARS-CoV-2 , Western Blotting , Eletroforese em Gel de Ágar/métodos , Eletroforese em Gel de Poliacrilamida , Géis , Humanos , Proteínas/química , Sefarose/química , Glicoproteína da Espícula de Coronavírus
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