Complete protease and phosphatase inhibitors

Whole cell extracts (WCE) were prepared in RIPA buffer (Sigma-Aldrich) with added phosphatase and complete protease inhibitors (Roche, Indianapolis, IN, USA). Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA, USA). 40 or 45 µg of WCE protein were electrophoresed on 10% SDS-PAGE gels and electroblotted on PVDF membranes (Bio-Rad) for western blotting with the following antibodies: HNRNPA2B1 (B1 epitope-specific^{32 (link)}) antibody # 18941 from IBL America (Minneapolis, MN USA); GAPDH cat.# sc-365062 (Santa Cruz Biotechnology, Dallas, TX, USA); β-actin (cat. # A5316, Sigma-Aldrich). Bands were visualized using a Bio Rad ChemiDoc MP imager and quantified by UN-SCAN-IT Graph Digitizer Software 7.1 (Silk Scientific, Orem, UT, USA).

Immunoprecipitations were performed as previously described [27 (link)]. Briefly, cells (5 × 10⁶) were lysed on ice for 15 minutes in lysis buffer [50mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP40] with phosphatase and complete protease inhibitors (Roche, Indianapolis, IN). Five percent of the total protein lysate (approximately 12.5 μg of 250 μg) was saved as an immunoprecipitation input loading control, then the remaining lysate was divided into equal volumes for overnight incubation at 4 °C with 1 μg of an antibody to STAT3 (sc-482, Santa Cruz Biotechnology, Inc., Dallas, TX), GRN (40-3400, Invitrogen, Grand Island, NY), or isotype control IgG (Caltag, Burlingame, CA). The lysate-antibody mixture was incubated for 4 hours at 4 °C with protein A/G PLUS agarose beads (Santa Cruz) blocked with 1% bovine serum albumin (Sigma-Aldrich) for 1 hour at 4 °C. The antibody-bead mixture was washed 3 times in lysis buffer, and eluted in sample buffer with10% β-mercaptoethanol.

Cell monolayers were washed twice with ice-cold PBS and lysed directly in ice-cold RIPA buffer (Pierce) containing a phosphatase and complete protease inhibitors (Roche) and a total of 30 μg protein of each sample run on 10% SDS-PAGE gels, and blotted onto nitrocellulose or PVDF membranes as described^{57 (link)}. Membranes were blocked in TBS-T (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) containing 5% (w/v) fat-free milk for 1–2 h and incubated with primary antibodies at the concentration indicated (Table S2) in TBS-T containing 5% BSA overnight at 4 °C. After washing in TBS-T (4 × 10 min), the blots were incubated for 1 h with a 1:10,000 dilution of the appropriate horseradish peroxidase-conjugated antibody (Cell Signalling Technologies) in TBS-T containing 5% (w/v) fat-free milk for 1 h at room temperature and proteins detected by enhanced chemiluminescence. Following the detection of phosphorylated Akt, or FOXO1 membranes were stripped in NaOH (200 mM) for 20 min at room temperature and re-probed for total Akt or FOXO1 protein respectively and β-actin, or α/β tubulin used as a loading control.

Cells were lysed in cold lysis buffer using the aforementioned lysis buffer with complete protease and phosphatase inhibitors (Roche Applied Science). Precleared cell lysate was incubated with anti-Flag or anti-p-MED1 antibodies overnight and then with ultralink immobilized protein A/G plus beads (Thermo Scientific) for 24 hours. Beads were washed four times with cold lysis buffer, and then samples were degenerated by boiling. Lastly, immune complexes were applied in Western blotting as previously described.