Journal: iScience
Article Title: CD9 exacerbates pathological cardiac hypertrophy through regulating GP130/STAT3 signaling pathway
doi: 10.1016/j.isci.2023.108070
Figure Lengend Snippet: CD9 Promotes Cardiac Hypertrophy by Regulating GP130 to Activate STAT3 (A and B) Immunoprecipitation and Western blot assays used anti-CD9 and anti-GP130 antibodies to detect GP130 binding with CD9 in cardiomyocytes, with IgG as control (n = 1). (C) Cardiomyocytes transfected with Ad CD9-shRNA or Ad Scr-shRNA underwent cycloheximide treatment (CHX). GP130 protein abundance analyzed through Western blot over time (0, 0.5, 1, and 2 h), quantified below (n = 3). (D) Effects of lysosome inhibitor CQ and proteasome inhibitor MG132 on GP130 expression in cardiomyocytes transfected with Ad CD9-shRNA or Ad Scr-shRNA examined (n = 3). (E) Cardiomyocytes transfected with Ad CD9-shRNA or Ad Scr-shRNA underwent co-IP of GP130, followed by immunoblotting of precipitates for ubiquitin (n = 3). (F) Cardiomyocytes transfected with Ad CD9 or Ad Control co-IP’d for GP130, followed by immunoblotting of precipitates for ubiquitin (n = 3). (G and H) Western blot assays performed on cardiomyocytes with CD9 knockdown or overexpression under Ang II stimulation, assessing CD9, GP130, p-STAT3, and STAT3 expression. GAPDH was loading control, with quantification below (n = 3). (I) Western blot nuclear plasma separation analysis of p-STAT3 nuclear translocation in cardiomyocytes transfected with Ad CD9-shRNA or Ad Scr-shRNA with or without Ang II stimulation (n = 3). (J) Representative immunofluorescence image showed p-STAT3 nuclear translocation in cardiomyocytes transfected with Ad CD9-shRNA or Ad Scr-shRNA with Ang II stimulation. Scale bar: 50 μm. (K and L) C57 mice injected with AAV CD9-shRNA or AAV CD9 two weeks pre-TAC surgery, sacrificed 4 weeks post-surgery. Western blot assays on cardiac tissues assessed CD9, GP130, p-STAT3, and STAT3 expression. GAPDH was the loading control, with quantification in the right panel (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
Article Snippet: Rabbit polyclonal ubiquitin anti-ub , Proteintech , Cat# 10201-2-AP; RRID: AB_671515.
Techniques: Immunoprecipitation, Western Blot, Binding Assay, Control, Transfection, shRNA, Quantitative Proteomics, Expressing, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Knockdown, Over Expression, Clinical Proteomics, Translocation Assay, Immunofluorescence, Injection
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