Mouse monoclonal anti myc tag 9b11

Antibodies used were: mouse monoclonal anti-Myc tag (9b11) (Cell Signaling #2276) used at 1:1000 for immunoblot analysis, 1:400 for immunostainings and 1:500 for immunoprecipitation, rat monoclonal anti-HA (3F10) (Roche #11867423001) used at 1:1000 for immunoblot analysis and 1:400 for immunostainings, rabbit anti-pS6K (Thr389) (Cell Signaling #9205) used at 1:1000 for immunoblot analysis, rabbit monoclonal anti-ERK1/2 (Cell Signaling #4695) used at 1:2000 for immunoblot analysis.

Whole cell extracts were obtained by disruption in modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% IGEPAL, protease and phosphatase inhibitors (Merck) and incubated at 4°C for 20 min, followed by centrifugation at 15200 rpm for 10 min. Proteins (20–30 μg) were separated into 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Membranes were blocked with 5% (wt/vol) nonfat powdered milk in TBS-T (20 mM Tris, pH 7.4; 100 mM NaCl; 1% Tween-20) and incubated with the following primary antibodies and dilutions: mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank) at 1:10; mouse monoclonal anti-myc-tag (9B11) (Cell Signaling) at 1:1000; rabbit monoclonal anti-GFP (E385) (Abcam) at 1:10000, mouse monoclonal anti-GAPDH (EMD Millipore) at 1:10000; rabbit MultiMab^™ anti-phospho-CK2 Substrate (pS/pT)DXE (Cell Signaling) at 1:1000. Anti-mouse IgG and anti-rabbit IgG HRP-conjugated secondary antibodies (Cell Signaling) were used at 1:5000, and HRP activity was visualized using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Relative densitometry was measured using ImageJ software.