Western Blotting (immunoblotting): History, Theory, Uses, Protocol and Problems

Sule, Rasheed; Rivera, Gabriela; Gomes, Aldrin V.

doi:10.2144/btn-2022-0034

Cited by 21 publications

(6 citation statements)

References 89 publications

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“…Conventional western blotting was performed following a standard protocol [ 20 ]. The following antibodies and dilutions were applied: anti-UCHL1 (1: 1000, 14730-1-AP/66230-1-Ig, Proteintech), anti-KLF5 (1:1000, 66850-1-Ig/21017-1-AP, Proteintech), anti-Ubiquitin (anti-Ub, 1:1000, 10201-2-AP, Proteintech), anti-Flag tag (1:1000, 66008-4-Ig, Proteintech), anti-Myc tag (1:1000, 60003-2-Ig, Proteintech), anti-TET1 (1:1000, 61,443, Proteintech, Wuhan, China), anti-TET3 (1: 1000, ABE290, Merck, Darmstadt, Germany) and anti-β-actin (1:2000, 20536-1-AP, Proteintech).…”

Section: Methodsmentioning

confidence: 99%

UCHL1 contributes to insensitivity to endocrine therapy in triple-negative breast cancer by deubiquitinating and stabilizing KLF5

Li,

Liang,

Zhou

et al. 2024

Breast Cancer Res

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Background Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that regulates ERα expression in triple-negative cancer (TNBC). This study aimed to explore the deubiquitination substrates of UCHL1 related to endocrine therapeutic responses and the mechanisms of UCHL1 dysregulation in TNBC. Methods Bioinformatics analysis was conducted using online open databases. TNBC representative MDA-MB-468 and SUM149 cells were used for in vitro and in-vivo studies. Co-immunoprecipitation was used to explore the interaction between UCHL1 and KLF5 and UCHL1-mediated KIF5 deubiquitination. CCK-8, colony formation and animal studies were performed to assess endocrine therapy responses. The regulatory effect of TET1/3 on UCHL1 promoter methylation and transcription was performed by Bisulfite sequencing PCR and ChIP-qPCR. Results UCHL1 interacts with KLF5 and stabilizes KLF5 by reducing its polyubiquitination and proteasomal degradation. The UCHL1-KLF5 axis collaboratively upregulates EGFR expression while downregulating ESR1 expression at both mRNA and protein levels in TNBC. UCHL1 knockdown slows the proliferation of TNBC cells and sensitizes the tumor cells to Tamoxifen and Fulvestrant. KLF5 overexpression partially reverses these trends. Both TET1 and TET3 can bind to the UCHL1 promoter region, reducing methylation of associated CpG sites and enhancing UCHL1 transcription in TNBC cell lines. Additionally, TET1 and TET3 elevates KLF5 protein level in a UCHL1-dependent manner. Conclusion UCHL1 plays a pivotal role in TNBC by deubiquitinating and stabilizing KLF5, contributing to endocrine therapy resistance. TET1 and TET3 promote UCHL1 transcription through promoter demethylation and maintain KLF5 protein level in a UCHL1-dependent manner, implying their potential as therapeutic targets in TNBC.

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Section: Methodsmentioning

confidence: 99%

UCHL1 contributes to insensitivity to endocrine therapy in triple-negative breast cancer by deubiquitinating and stabilizing KLF5

Li,

Liang,

Zhou

et al. 2024

Breast Cancer Res

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“…Western blotting was performed for IPO9, RAP2A, DDX17, and GAPDH, as described previously ( Table 2 ). Enhanced chemiluminescence reagents were used to detect protein expression, and quantitative analysis was conducted using Image J software ( 28 ).…”

Section: Methodsmentioning

confidence: 99%

Elucidating sleep disorders: a comprehensive bioinformatics analysis of functional gene sets and hub genes

Lin,

Liu,

2024

Front. Immunol.

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BackgroundSleep disorders (SD) are known to have a profound impact on human health and quality of life although their exact pathogenic mechanisms remain poorly understood. MethodsThe study first accessed SD datasets from the GEO and identified DEGs. These DEGs were then subjected to gene set enrichment analysis. Several advanced techniques, including the RF, SVM-RFE, PPI networks, and LASSO methodologies, were utilized to identify hub genes closely associated with SD. Additionally, the ssGSEA approach was employed to analyze immune cell infiltration and functional gene set scores in SD. DEGs were also scrutinized in relation to miRNA, and the DGIdb database was used to explore potential pharmacological treatments for SD. Furthermore, in an SD murine model, the expression levels of these hub genes were confirmed through RT-qPCR and Western Blot analyses.ResultsThe findings of the study indicate that DEGs are significantly enriched in functions and pathways related to immune cell activity, stress response, and neural system regulation. The analysis of immunoinfiltration demonstrated a marked elevation in the levels of Activated CD4+ T cells and CD8+ T cells in the SD cohort, accompanied by a notable rise in Central memory CD4 T cells, Central memory CD8 T cells, and Natural killer T cells. Using machine learning algorithms, the study also identified hub genes closely associated with SD, including IPO9, RAP2A, DDX17, MBNL2, PIK3AP1, and ZNF385A. Based on these genes, an SD diagnostic model was constructed and its efficacy validated across multiple datasets. In the SD murine model, the mRNA and protein expressions of these 6 hub genes were found to be consistent with the results of the bioinformatics analysis.ConclusionIn conclusion, this study identified 6 genes closely linked to SD, which may play pivotal roles in neural system development, the immune microenvironment, and inflammatory responses. Additionally, the key gene-based SD diagnostic model constructed in this study, validated on multiple datasets showed a high degree of reliability and accuracy, predicting its wide potential for clinical applications. However, limited by the range of data sources and sample size, this may affect the generalizability of the results.

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“…P-Tau181 in the serum of neurodegenerative disorders is 1.81 pg/mL, and ORF 1p in carcinomas and high-risk precursors during carcinogenesis is 10 –17 M . Conventional techniques, including ELISA, Western blot, and mass spectrometry, are not able to quantify these rare biomarkers. A digital immunoassay (DIA) is a type of immunoassay known for its ultrahigh sensitivity and specificity .…”

Section: Introductionmentioning

confidence: 99%

Amplification-Free Digital Immunoassay down to the Attomolar Level by Synergistic Sedimentation of Brownian Motion Suppression and Dehydration Transfer

Zhang,

Chai,

Pan

et al. 2024

Anal. Chem.

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Amplification-free digital immunoassays (DIAs) typically utilize optical nanoparticles to enhance single immunocomplex molecule detection. The efficiency and uniformity of transferring the nanoparticles from a bulk solution to a solid surface determine the limit of detection (LOD) and the accuracy of DIAs. Previous methods suffer from issues like low efficiency, nonuniform distribution, and particle aggregation. Here, we present a novel technique named synergistic sedimentation of Brownian motion suppression and dehydration transfer (SynSed) for nanoparticles using water-soluble polymers. The efficiency of transferring quantum dots (QDs) was increased from 10.7 to 91.4%, and the variation in QD distribution was restricted to 8.8%. By incorporating SynSed into DIAs, we achieved a remarkable reduction in the LOD (down to 3.9 aM) for carcinoembryonic antigen and expanded the dynamic range to cover 3 orders of magnitude in concentration, ranging from 0.01 to 10 fM. DIAs enhanced with SynSed possess ultrahigh sensitivity, advanced accuracy, and specificity, offering a great premise in early disease diagnostics, risk stratification, and treatment response monitoring.

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Western Blotting (immunoblotting): History, Theory, Uses, Protocol and Problems

Cited by 21 publications

References 89 publications

UCHL1 contributes to insensitivity to endocrine therapy in triple-negative breast cancer by deubiquitinating and stabilizing KLF5

UCHL1 contributes to insensitivity to endocrine therapy in triple-negative breast cancer by deubiquitinating and stabilizing KLF5

Elucidating sleep disorders: a comprehensive bioinformatics analysis of functional gene sets and hub genes

Amplification-Free Digital Immunoassay down to the Attomolar Level by Synergistic Sedimentation of Brownian Motion Suppression and Dehydration Transfer

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