Anti gsk 3α β sc 7291

L-BMAA, URB597, anti-ANP32e (SAB2100124), iodoacetamide, amoniumbicarbonate and urea were purchased from Sigma Aldrich. Anti-GSK-3α/β (sc-7291) and anti-GAPDH (sc-32233) and anti-Sec61a (sc-12322) were from Santa Cruz. Sequence grade trypsin was from Promega. Acetonitrile (ACN), water and trifluoretic acid (TFA) were all HPLC grade and purchased from Sigma Aldrich.

To prepare protein lysate, tumors and PDX cell lines PCB82 and PCB380 treated with vehicle (control) and tideglusib were lysed in radio immunoprecipitation (RIPA) buffer (89901, Thermo Fisher Scientific) containing both protease and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO). Lysates were homogenized and clarified by centrifugation at 14,000 rpm for 10 minutes. Thirty μg of protein were electrophoresed in 7.5-10% mini protean polyacrylamide gel (4561024, Bio-Rad, Hercules, CA, USA) transferred to PVDF membranes (1620255, Bio-Rad) for immunoblot analysis with anti-GSK3α/β (sc-7291, Santa Cruz Biotechnology, Dallas, TX, USA) (the monoclonal antibody used is raised against the amino acid 1-420 representing the full length GSK3β), anti-Phos-β-Catenin (9561, Cell Signaling, Danvers, MA, USA), anti-β-Catenin (9562, Cell Signaling), anti-MHC (MAB4470, R&D systems, Minneapolis, MN, USA), anti-myogenin (sc-576, Santa Cruz Biotechnology), anti-β-actin (ab8227, Abcam, San Francisco, CA, USA). Blots were developed using FluorChem Q system (92-14095-00, protein simple, San Jose, CA, USA).