Anti mef2c sc 13266

C2C12 cells were cultured on Thermonax plastic cover slips. Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA). Fixed cells were blocked and permeabilized with 0.1% Triton-X-100 in PBS supplemented with 10% horse serum. After blocking, cells were incubated with primary antibody in PBS with 10% horse serum. The next day, cells were washed with PBS and probed with secondary antibody conjugated to Alexa-Fluor 488 (Invitrogen) or Alexa-Fluor 633 (Invitrogen). Cells were washed with PBS thrice, and counter-stained with DAPI before being viewed under BX53 fluorescence microscope or FluoView FV1000 confocal fluorescence microscope (Olympus). Primary antibodies used for immunofluorescence are anti-Actn2 (A7811, Sigma-Aldrich; 1:500), and anti-MEF2C (sc-13266, Santa Cruz Biotechnology;1:200). Rhodamine-phalloidin (R415, Life Technologies; 1:200) was used for staining filamentous actin (F-actin).

Cells were lysed in Protein extraction buffer (50 mM Tris pH 8, 150 mM NaCl, 0.5% NP40 and 1 mM EGTA) and protein concentrations were determined using the Bio-Rad DC Protein Assay reagents package (Bio-rad). A total of 40 µg of total proteins were separated by SDS-PAGE and transferred to Nitrocellulose membranes, blocked 1 h at room temperature (10% milk, 0.1% Tween-20 TBS) and probed at 4 °C overnight (2% milk, 0.1% Tween-20 TBS) with the following primary antibodies: anti-CBP (sc-369, Santa Cruz Biotechnology); anti-P300 (sc-585, Santa Cruz Biotechnology); anti-Myf5 (sc-302, Santa Cruz Biotechnology); anti-Myogenin (sc-576, Santa Cruz Biotechnology); anti-Mef2C (sc-13266, Santa Cruz Biotechnology); anti-Vinculin (sc-5573, Santa Cruz Biotechnology) and anti-MHC (MF20, Developmental Studies Hybridoma Bank). After washings, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. Detection was performed using Lumi-light^plus Western Blotting substrate (Roche) according to manufacturer’s instruction. Original scans of all western blots shown in this study can be found in Supplementary Fig. S7.