Application of Proteomics Technology Based on LC-MS Combined with Western Blotting and Co-IP in Antiviral Innate Immunity.

As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.

Improving rigor and reproducibility in western blot experiments with the blotRig analysis.

Western blot is a popular biomolecular analysis method for measuring the relative quantities of independent proteins in complex biological samples. However, variability in quantitative western blot data analysis poses a challenge in designing reproducible experiments. The lack of rigorous quantitative approaches in current western blot statistical methodology may result in irreproducible inferences. Here we describe best practices for the design and analysis of western blot experiments, with examples and demonstrations of how different analytical approaches can lead to widely varying outcomes. To facilitate best practices, we have developed the blotRig tool for designing and analyzing western blot experiments to improve their rigor and reproducibility. The blotRig application includes functions for counterbalancing experimental design by lane position, batch management across gels, and analytics with covariates and random effects.

Methods for Monitoring ER-Phagy.

The endoplasmic reticulum (ER) serves as a central hub for protein synthesis, folding, and lipid biosynthesis in eukaryotic cells. Maintaining ER homeostasis is essential for optimal cellular function, and one mechanism that has garnered attention is endoplasmic reticulum-specific autophagy, or ER-phagy. ER-phagy selectively removes specific ER portions, playing a pivotal role in cellular health and adaptation to environmental stressors. ER-phagy can be induced by diverse cellular conditions such as amino acid starvation, disruption of ER quality control mechanisms, and accumulation of misfolded ER protein, highlighting cellular adaptability and the significance of ER-phagy in stress responses. Clinically relevant mutations in ER-phagy receptors are implicated in various diseases, underlining the fundamental importance of ER-phagy in ER homeostasis. Here, we provide comprehensive protocols and general considerations while investigating ER-phagy using three fundamental techniques-Western blotting, immunofluorescence, and flow cytometry-commonly used in ER-phagy detection and quantitation.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Detection of Plasmodium knowlesi in whole blood samples with sandwich enzyme-linked immunosorbent assay (ELISA) using rhoptry-associated protein 1 specific polyclonal antibodies.

BACKGROUND OBJECTIVES: Plasmodium knowlesi, a simian malaria species, is now known to infect humans. Due to disadvantages in the current diagnosis methods, many efforts have been placed into developing new methods to diagnose the disease. This study assessed the ability of the PkRAP-1 sandwich enzyme-linked immunosorbent (ELISA) to detect P knowlesi antigens in whole blood specimens. METHODS: Western blot assay was conducted to evaluate the ability of raised mouse and rabbit anti-PkRAP-1 polyclonal antibodies to bind to the native proteins in P. knowlesi lysate. The polyclonal antibodies were then used in sandwich ELISA to detect P. knowlesi. In the sandwich ELISA, mouse and rabbit polyclonal antibodies were used as the capture and detection antibodies, respectively. The limit of detection (LOD) of the assay was determined using P. knowlesi A1H1 culture and purified recombinant PkRAP-1. RESULTS: Western blot results showed positive reactions towards the proteins in P. knowlesi lysate. The LOD of the assay from three technical replicates was 0.068% parasitaemia. The assay performance in detecting P. knowlesi was 83% sensitivity and 70% specificity with positive and negative predictive values of 74% and 80%, respectively. The anti-PkRAP-1 polyclonal antibodies did not cross-react with P. falciparum and healthy samples, but P. vivax by detecting all 12 samples. INTERPRETATION CONCLUSION: PkRAP-1 has the potential as a biomarker for the development of a new diagnostic tool for P. knowlesi detection. Further studies need to be conducted to establish the full potential of the usage of anti-PkRAP-1 antibodies for P. knowlesi detection.

Rapid tests should be used with caution for HIV-1 primary infection screening.

Rapid tests allow outpatient, low cost, reliable, screening for chronic HIV infection. However, data regarding their sensitivity on primary infection remain scarce. The objective of this study was to assess sensitivity of nine HIV rapid tests for primary HIV-1 infection screening. Seventy-five serum samples from patients during HIV-1 primary infection were included. Primary infection was diagnosed by a positive 4th generation ELISA and HIV-1 RNA positivity confirmed by Western blot patterns associated with HIV-1 primary infection. Early seroconversion was defined as the absence of antibodies on HIV-1 Western blot associated with HIV-1 RNA and p24-antigen positivity. An identical sensitivity (95% CI) of 76.7% (65.2-84.2%) was observed for HIV 1/2 STAT-PAK® Assay (STAT-PAK), INSTI™ HIV-1/HIV-2 antibody Test (INSTI), SURE CHECK® HIV 1/2 (SURE CHECK) and MULTISURE HIV rapid test (MULTISURE) with visual reading. Sensitivity was 74.7% (63.8-83.1%) for MULTISURE (automatic reading), 77.0% (66.3-85.1%) for FIRST RESPONSE® Test VIH 1-2.O CARTE (FIRST RESPONSE), 83.8% (73.8-90.5%) for VIKIA HIV1/2® (VIKIA), 88.0% (78.7-93.6%) for Genie™ Fast HIV 1/2 (Genie Fast), 88.6% (79.0-94.1%) for Hexagon HIV (Hexagon), and 92.8% (83.6-96.3%) for Exacto® TEST HIV Pro (Exacto). However, rapid tests performed poorly for the early seroconversion subgroup (n = 14), with sensitivities ranging from 7% (1.3-31.5%) for STAT-PAK, INSTI, SURE CHECK, MULTISURE (automatic reading), to 29% (12-55%) for FIRST RESPONSE, 31% (13-58%) for VIKIA, 43% (21-67%) for Hexagon and 57.1% (32.6-78.6%) for Exacto and Genie Fast. Overall, despite significant discrepancies in sensitivity, HIV rapid tests should be used with caution in the context of a suspected primary infection.

Protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step immunoprecipitation approach.

Co-immunoprecipitation (coIP) is an experimental technique to study protein-protein interactions (PPIs). However, single-step coIP can only be used to identify the interaction between two proteins and does not solve the interaction testing of ternary complexes. Here, we present a protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step coIP approach. We describe steps for cell culture and transfection, elution of target proteins, and two-step coIP including western blot analyses. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Protocol for identifying OXCT1-mediated LACTB succinylation sites in vitro.

OXCT1 acts as a succinyltransferase to promote serine beta-lactamase-like protein (LACTB) K284 succinylation. Here, we present a protocol for detecting OXCT1-mediated LACTB succinylation levels and sites. We describe steps for using western blotting (WB) and mass spectrometry to determine OXCT1-mediated LACTB succinylation levels and sites in vitro. This protocol can be applied to detect and identify succinylation levels and sites on other proteins. For complete details on the use and execution of this protocol, please refer to Ma et al.1.

Biochemical Detection of Capsid in the Nucleus During HIV-1 Infection.

Recent evidence has shown that uncoating and reverse transcription precede nuclear import. These recent breakthroughs have been made possible through the development of innovative biochemical and imaging techniques. This method outlines the biochemical assay used for detecting the presence of the HIV-1 core in the nuclear compartment. In this procedure, human cells are infected with HIV-1NL4-3, with or without the inclusion of PF74, a small molecule that inhibits core entry into the nuclear compartment. Subsequently, cells are separated into cytosolic and nuclear fractions. To assess whether the capsid protein has reached the nuclear compartment, cytosolic and nuclear fractions are subjected to Western blot analysis, utilizing antibodies specific to the HIV-1 capsid protein p24. To validate the true origin of these fractions, Western blot analysis employing antibodies against cytosolic and nuclear markers are also performed. In summary, this assay provides a reliable and efficient means to detect the presence of the HIV-1 capsid protein in the nucleus during infection under various conditions.

Chemical cross-linking to study protein self-assembly in cellulo.

Many proteins self-assemble into dimers and higher-order oligomers. Therefore, the goal of this protocol is to characterize the conformational states of an endogenous protein of interest. Here, we present a protocol for assessing protein self-assembly in cell lysates using chemical cross-linking. We describe steps for chemical cross-linking with recombinant proteins as well as steps for cell culture and cell lysate preparation, chemical cross-linking, SDS-PAGE, and western blotting for the detection of endogenous proteins. For complete details on the use and execution of this protocol, please refer to Balaji et al.1.

Validation of a novel western blot assay to monitor patterns and levels of alpha dystroglycan in skeletal muscle of patients with limb girdle muscular dystrophies.

The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics.

A Multiplexed Approach to Assess Small Cell Lung Cancer Subtype Heterogeneity in Primary and Patient-Derived Tumor Samples.

Unlocking the heterogeneity of cancers is crucial for developing therapeutic approaches that effectively eradicate disease. As our understanding of markers specific to cancer subclones or subtypes expands, there is a growing demand for advanced technologies that enable the simultaneous investigation of multiple targets within an individual tumor sample. Indeed, multiplex approaches offer distinct benefits, particularly when tumor specimens are small and scarce. Here we describe the utility of two fluorescence-based multiplex approaches; fluorescent Western blots, and multiplex immunohistochemistry (Opal™) staining to interrogate heterogeneity, using small cell lung cancer as an example. Critically, the coupling of Opal™ staining with advanced image quantitation, permits the dissection of cancer cell phenotypes at a single cell level. These approaches can be applied to patient biopsies and/or patient-derived xenograft (PDX) models and serve as powerful methodologies for assessing tumor cell heterogeneity in response to therapy or between metastatic lesions across diverse tissue sites.

Assessing Protein Expression in Patient-Derived Xenografts Using Western Blotting.

The use of patient-derived xenografts (PDXs) in cancer research is increasing due to their ability to closely mimic the features of patient tumors. The ability to quickly and robustly measure protein expression levels in these tissues is a key methodology required in a broad range of experimental designs. Western blotting (WB) is a cost effective and simple tool that is highly specific and sensitive for detecting and quantifying individual proteins, posttranslational modifications and aberrant signaling pathways. Here, we described a method to assess protein expression in PDX tissues using WB to detect proteins involved in cell growth signaling pathways.

Photosensitive Hydrogel with Temperature-Controlled Reversible Nano-Apertures for Single-Cell Protein Analysis.

Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in-gel probing and separation resolution. Here, a highly sensitive temperature-controlled single-cell western blotting (tc-scWB) method is introduced, which is based on a thermo/photo-dualistic-sensitive polyacrylamide hydrogel, namely acrylic acid-functionalized graphene oxide (AFGO) assisted, N-isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high-temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low-temperature for better antibody penetration into the hydrogel. The tc-scWB method enables the separation and profiling of small-molecule-weight proteins with highly crosslinked gel (12% T) in SDS-PAGE. The tc-scWB is demonstrated on three metabolic and ER stress-specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc-scWB is a general toolbox for the analysis of low-abundance small-molecular functional proteins at the single-cell level.

An efficient method for detecting membrane protein oligomerization and complex using 05SAR-PAGE.

Oligomerization is an important feature of proteins, which gives a defined quaternary structure to complete the biological functions. Although frequently observed in membrane proteins, characterizing the oligomerization state remains complicated and time-consuming. In this study, 0.05% (w/v) sarkosyl-polyacrylamide gel electrophoresis (05SAR-PAGE) was used to identify the oligomer states of the membrane proteins CpxA, EnvZ, and Ma-Mscl with high sensitivity. Furthermore, two-dimensional electrophoresis (05SAR/sodium dodecyl sulfate-PAGE) combined with western blotting and liquid chromatography-tandem mass spectrometry was successfully applied to study the complex of CpxA/OmpA in cell lysate. The results indicated that 05SAR-PAGE is an efficient, economical, and practical gel method that can be widely used for the identification of membrane protein oligomerization and the analysis of weak protein interactions.

An optimized Western blot assay provides a comprehensive assessment of the physiological endoproteolytic processing of the prion protein.

The prion protein (PrPC) is subjected to several conserved endoproteolytic events producing bioactive fragments that are of increasing interest for their physiological functions and their implication in the pathogenesis of prion diseases and other neurodegenerative diseases. However, systematic and comprehensive investigations on the full spectrum of PrPC proteoforms have been hampered by the lack of methods able to identify all PrPC-derived proteoforms. Building on previous knowledge of PrPC endoproteolytic processing, we thus developed an optimized Western blot assay able to obtain the maximum information about PrPC constitutive processing and the relative abundance of PrPC proteoforms in a complex biological sample. This approach led to the concurrent identification of the whole spectrum of known endoproteolytic-derived PrPC proteoforms in brain homogenates, including C-terminal, N-terminal and, most importantly, shed PrPC-derived fragments. Endoproteolytic processing of PrPC was remarkably similar in the brain of widely used wild type and transgenic rodent models, with α-cleavage-derived C1 representing the most abundant proteoform and ADAM10-mediated shedding being an unexpectedly prominent proteolytic event. Interestingly, the relative amount of shed PrPC was higher in WT mice than in most other models. Our results indicate that constitutive endoproteolytic processing of PrPC is not affected by PrPC overexpression or host factors other than PrPC but can be impacted by PrPC primary structure. Finally, this method represents a crucial step in gaining insight into pathophysiological roles, biomarker suitability, and therapeutic potential of shed PrPC and for a comprehensive appraisal of PrPC proteoforms in therapies, drug screening, or in the progression of neurodegenerative diseases.

Pre-treatment of the beta3-adrenergic receptor agonist BRL37344 reduces in vivo myocardial ischemia/reperfusion injury by improving AMPK and SIRT1 activity and by suppressing mTOR and p70S6K signaling pathways

Abstract This study aimed to investigate the role and signaling pathways of β3-AR in myocardial ischemia/reperfusion (I/R) injury, which is one of the leading causes of death worldwide. 47 male rats were randomly divided into two main groups to evaluate infarct size and molecular parameters. Rats in both groups were randomly divided into 4 groups. Control (sham), I/R (30 min ischemia/120 min reperfusion), BRL37344 (BRL) (A) (5 µg/kg single-dose pre-treatment (preT) before I/R) and BRL (B) (5 µg/kg/day preT for 10 days before I/R). Infarct size was determined with triphenyltetrazolium chloride staining and analyzed with ImageJ program. The levels of AMPK, SIRT1, mTOR, and p70SK6 responsible for cellular energy and autophagy were evaluated by western blot. Infarct size increased in the I/R group (44.84 ± 1.47%) and reduced in the single-dose and 10-day BRL-treated groups (32.22 ± 1.57%, 29.65 ± 0.55%; respectively). AMPK and SIRT1 levels were decreased by I/R but improved in the treatment groups. While mTOR and p70S6K levels increased in the I/R group, they decreased with BRL preT. BRL preT protects the heart against I/R injury. These beneficial effects are mediated in part by activation of AMPK and SIRT1, inhibition of mTOR and p70S6K, and consequently protected autophagy.

Effects of the microRNA-99a-5p/VLDLR axis in lung cancer cell sensitivity to chemotherapy and its mechanism

Abstract Lung cancer is a major cause of cancer-related death worldwide. This study investigated the regulatory effects of the microRNA-99a-5p (miR-99a-5)/VLDLR axis on lung cancer cell sensitivity to chemotherapy and its mechanism. miR-99a-5p and VLDLR expression levels were quantified using RT-qPCR and western blotting, respectively. The IC50 value of cisplatin (DDP) was determined using a CCK-8 assay. Lung cancer cell proliferation and apoptosis were measured using the CCK-8 assay and flow cytometry, respectively. The mRNA expression levels of apoptosis-related factors (Bax, Bcl-2, and Caspase-3) were evaluated using RT-qPCR. The direct relationship between miR-99a-5p and VLDLR was validated using dual-luciferase reporter gene and RIP assays. miR-99a-5p was weakly expressed in DDP-resistant lung cancer cells. Overexpression of miR-99a-5p promoted DDP sensitivity, suppressed proliferation and colony formation, and promoted apoptosis of A549/DDP cells in vitro. Mechanistically, miR-99a-5p restrained VLDLR expression by binding to VLDLR 3'UTR, and miR-99a-5p mediated inhibition of VLDLR regulated the DDP sensitivity, proliferation, and apoptosis of A549/ DDP cells. Overexpression of miR-99a-5p inhibited the growth of A549 cells and increased chemosensitivity of A549 cells to DDP in vivo. In conclusion, miR-99a-5p overexpression promotes sensitivity to DDP and cell apoptosis by downregulating VLDLR expression in A549/ DDP cells.

Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots.

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs' versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson's-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression.

Confirmation value of Western blotting in detecting anti-treponema pallidum specific antibodies with suspicious results.

BACKGROUND: Due to the inconsistent results of anti-treponema pallidum (TP) specific antibodies by enzyme-linked immunosorbent assay (ELISA) and Treponema pallidum granule agglutination assay (TPPA) in clinical work, there will be a certain proportion of false-positives and false-negatives depending on TPPA as confirmation results. This study aimed to evaluate the necessity of Western blotting (WB) in samples with inconsistent results in detecting anti-TP antibodies by ELISA and TPPA. METHODS: Specific anti-TP test results in our clinical laboratory were retrospectively analyzed. The specimens with a positive or a negative result, but with colored ELISA plates, were retested by TPPA. WB was used to confirm the suspicious results between ELISA and TPPA. The Chi-square test was used to analyze whether the difference was statistically significant. RESULTS: A total of 106,757 anti-TP specimens were screened by ELISA from August 2018 to December 2019; 3972 were retested by TPPA, and 3809 were positive by TPPA. ELISA and TPPA showed different results in 163 specimens. Among them, 29 specimens were negative and 134 were positive by ELISA; 76 were negative, 23 were positive, and 64 were "reserve" by TPPA; 93 were negative, 31 were positive, and 39 were suspicious by the WB confirmation test. Compared with WB, the difference in the results of ELISA and TPPA was statistically significant. CONCLUSIONS: TPPA is an effective retest method for anti-TP antibody detection. If the results of anti-TP antibodies by ELISA and TPPA are inconsistent, it is necessary to use WB for confirmation. Trial registration This retrospective analysis is in accordance with the ethical guidelines of China and approved by the second hospital of Jiaxing (jxey-2018048).