Western Blot and Protein Immunoprecipitation

For Western blot, cells were lysed in cold radioimmunoprecipitation buffer (50 mM Tris–HCl pH 7.5, 1% NP-40, 150 mM NaCl, 5 mM EDTA, 0.25% NaDOC, and 0.1% SDS) supplemented with protease and phosphatase inhibitors, centrifuged at 14,000 rpm for 20 min at 4 °C, and supernatant was collected as whole cell extract. 40 μg of proteins were resolved by SDS-polyacrylamide electrophoresis in 10% to 12% gels and transferred by electroblotting to a nitrocellulose membrane.
For protein immunoprecipitation and coimmunoprecipitation experiments, cells were lysed as already reported (46 (link)). Briefly, 500 μg whole cell extract was incubated overnight with 50 μl of protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, #sc-2003) and 2.5 μg of mouse anti-SOX2 (R&D System, #MAB2018), mouse anti-p38 (Santa Cruz Biotechnology, #81621), or normal mouse IgG (Santa Cruz Biotechnology, #sc-2025). Proteins were eluted after boiling for 10 min at 95 °C in 2× Laemmli sample buffer (Bio-Rad, catalog no.: 1610747) and detected by using SuperSignal West Femto (ThermoFisher Scientific) and imaged with ChemiDocTM Imaging Systems (Bio-Rad). A list of primary antibodies used is reported in Table S5.