Anti myod sc 760

Cells (2 million cells/mL) were treated with 0.3% formaldehyde in medium for 10 min at 37 °C, mixed with 1.25 M glycine dissolved in PBS to a final concentration of 0.125 M, and incubated for 5 min at room temperature. The cells were then washed twice in cold PBS and pelleted. The pellet was resuspended in 1 ml of RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5 mM DTT, and 1 mM PMSF/cocktail) and incubated on ice with frequent vortexing for 30 min, and the lysate was obtained by centrifugation at 13,000 RPM for 10 min. Antibodies (anti-Ab81901, Abcam; anti-MyoD, sc760, Santa Cruz) were added and the samples were incubated for 4 h at 4 °C and washed twice in RIPA buffer, four times in 1 M RIPA buffer (50 mM Tris, pH 7.4, 1 M NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate), and twice in RIPA buffer, all in Handee spin columns (Pierce). The beads were resuspended in RIPA buffer and treated with proteinase K at 45 °C for 45 min. RNA samples were extracted with 1 ml TRIzol, and co-precipitated RNAs were purified with an RNeasy Mini Kit (QIAGEN) and detected by RT-qPCR.