Igg fc specific peroxidase

Whole-cell and tumour lysates were isolated using RIPA buffer supplemented with protease inhibitors (Complete Mini, Sigma-Aldrich Inc.) and phosphatase inhibitors including sodium pyrophosphate, β-glycerophosphate, sodium fluoride and sodium orthovanadate (Sigma-Aldrich). The following primary antibodies were used: anti-MCU (#D2Z3B 1:1000, Cell Signaling), anti-MICU1 (#HPA037479 1:1000, Sigma-Aldrich), anti-MICU2 (#ab101465 1:1000, Abcam), anti-phospho-SMAD3 (#C25A9, 1:1000, Cell Signaling), anti-SMAD3 (#9513, 1:1000, Cell Signaling), anti-MYOG (#sc-12732, 1:500, Santa-Cruz), anti-MHC (#sc-32732, 1:250, Santa-Cruz) anti-HSP60 (#611563, BD Biosciences), anti-phospho-NF-κB (#3037, 1:1000, Cell Signaling), anti-NF-κB (#ab52175, 1:500, Abcam), anti-phospho-p38 MAPK (#9211, 1:1000, Cell Signaling), anti-p38 MAPK (#9212, 1:1000, Cell Signaling), and anti-β-actin (#A2228, 1:10,000, Sigma-Aldrich). Appropriate secondary antibodies (IgG-Fc Specific-Peroxidase) of mouse or rabbit origin (Sigma-Aldrich) were used.

Cells were lysed with RIPA lysis buffer (50 mM Tris HCl pH 7.5, 40 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mM EDTA) containing protease inhibitor cocktail (Complete Mini, Sigma-Aldrich Inc.). Immunoblots were probed overnight with primary antibodies and incubated with relevant horseradish peroxidase (HRP)-conjugated secondary antibodies. The following primary antibodies were used: anti-BRD4 (short and long isoforms) (Cat. #: ab128874, Abcam (CB, UK), WB 1:1000), anti-BRD4 E2A7X (long isoform) (Cat. #: 13440S, CST (MA, USA), WB 1:1000), anti-GDF8/Myostatin (Cat. #: ab201954, Abcam, WB 1/1000), anti-MHC (Cat. #: sc-32732, Santa-Cruz Biotechnology Inc., WB 1:250), anti-MYOG (Cat. #: sc-12732, Santa-Cruz Biotechnology Inc., WB 1/250), anti-ITGA4 (Cat. #: 8440, CST, WB 1:1000), anti-ITGA5 (Cat. #: 4705, CST, WB 1:1000) and anti-β-actin (Cat. #: A2228, WB 1:10,000, Sigma-Aldrich Inc.), anti-Zeb2 (Cat. #: ab138222, Abcam, WB 1/1000) and anti-c-Myc (Cat. #: sc-40, Santa-Cruz Biotechnology Inc., WB 1/500). Appropriate secondary antibodies (IgG-Fc Specific-Peroxidase) of mouse or rabbit origin (Sigma-Aldrich Inc.) were used. Proteins were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Quantification of western blots was done with ImageJ (v1.53t) software. The signal was normalized to β-actin.

Cells were lysed using RIPA or SDS lysis buffer supplemented with protease inhibitors (Complete Mini, Sigma-Aldrich). The following primary antibodies were used: anti-EHMT2 (#3306S, 1:300, Cell Signaling), anti-MHC (#sc-32732, 1:300, Santa Cruz Biotechnology), anti-Myogenin (#sc-12732, 1:250, Santa Cruz Biotechnology), anti-DKK1 (#sc374574, 1:300, Santa Cruz Biotechnology), anti-active-ß-catenin (#05–665, 1:500, Merck Millipore), anti-H3K9me2 (#9753S, 1:1000, Cell Signaling), anti-ß-actin (#A2228, 1:10,000; Sigma-Aldrich), and anti-H3 (#ab1791, 1:10,000; Abcam). Appropriate secondary antibodies (IgG-Fc Specific-Peroxidase) of mouse or rabbit origin (Sigma Aldrich) were used.