Sensitive supersignal west femto maximum sensitivity substrate

Total cell lysates from human RMS cell lines and human myoblasts were obtained following lysis in RIPA lysis buffer supplemented with protease inhibitors (Roche). Western blot analysis was performed similar to Ignatius et. al., 2017 (27 (link)). Membranes were developed using an ECL reagent (Western Lightning Plus ECL, PerkinElmer; or sensitive SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific). Membranes were stripped, rinsed, and re-probed with the respective internal control antibodies. List of primary and secondary antibodies is included in supplementary data (Supplemental Table 1). Western blots were quantified using ImageJ software, normalizing proteins of interest to their respective β-tubulin expression and the control (either hSKMC or Scr of the same time point). If control expression was zero, quantifications shown are shown as a ratio of the protein of interest expression to its respective β-tubulin expression.

Total cell lysates from human rhabdomyosarcoma cell lines and human myoblasts were obtained following lysis in 2% SDS lysis buffer supplemented with protease inhibitors (Santa Cruz Biotechnology). Western blot analysis used primary antibodies: rabbit a-NOTCH1 (Abcam), a-Cleaved NOTCH1 (Cell Signaling Technology), a-SNAI1 goat pAB (R&D Systems), a-Myosin Heavy Chain mouse mAb (monoclonal antibody) myosin heavy chain (a-MF20, R&D), MEF2C rabbit mAb (CST); and secondary antibodies: HRP (horseradish peroxidase) anti-rabbit (CST, 7074) or HRP anti-mouse (GE Healthcare, NA93IV). Membranes were developed using an ECL reagent (Western Lightning Plus ECL, PerkinElmer; or sensitive SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific). Membranes were striped, rinsed, and re-probed with the respective internal control rabbit a-Lamin B1 (Abcam) or rabbit a-GAPDH (CST).