Myod1

Cells were lysed by RIPA buffer and added the bromophenol blue loading buffer, and then the samples were boiled for 10 min and centrifuged at 12,000 rpm for 5 min. The whole-cell lysate was separated into 8% SDS-acrylamide gels and transferred to PVDF membranes. After that, the membranes was blocks by 5% milk in TBST and probed with primary antibodies including mouse SETD3 (3B3, generated by Wuhan Dia-An Company), rabbit polyclonal SETD3 (Abclonal, A8071), MyoD1 (Proteintech, 18943-l-AP), HDAC1 (Abclonal, A2238), Cyclin E1 (Cell Signaling Technology, 20808 S), Myogenin (Abcam, ab124800; or Santa Cruz, D-10, sc-13137), MHC (Developmental Studies Hybridoma Bank, MF-20), β-Actin (Proteintech, 6008-I-Ig), and α-Tubulin (Sigma, T9026). For generation of mouse monoclonal SETD3 antibody, His-tagged full-length human SETD3 protein was expressed in E. Coli and purified as described previously^{14 (link)}. Purified His-SETD3 proteins were immunizated into 5-8 weeks old Balb/C mice and boosted additional 4 times. After several steps including hybridoma production, screening, cloning, and expanding the hybridomas, a subclone named 3B3 was validated and amplified followed the procedure described as before^{39 (link)}. Membranes were further probed with horseradish peroxidase (HRP)-conjugated secondary antibodies and the protein bands were visualized using chemiluminescence detection reagents.