Chemiluminescence based kit

Cell lysates were obtained using standard techniques and RIPA lysis buffer (Beyotime Institute of Biotechnology, Changzhou, China). For immunoprecipitation, lysates were incubated with IGF-IRβ (Zymed Laboratories, South San Francisco, CA, USA) antibody overnight at 4°C. Agarose beads conjugated with A/G were then added and incubated for 4 hours at 4°C. The immunocomplexes were spun, washed 3 times with cold phosphate buffered saline (PBS) and once with lysis buffer, and then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). For Western blotting (WB), proteins (50–80 μg) were electrophoresed on 6% to 12% SDS-PAGE then transferred to nitrocellulose membranes and probed with specific primary antibodies including IGF-IRβ (Zymed Laboratories, South San Francisco, CA, USA), pIGF-IR (Tyr1131), Phospho-Tyrosine, pAkt (Ser 473), Akt, pMAPK (Thr202/Tyr204), MAPK (Erk1/2), caspase-3, PARP (all from Cell Signaling Technology, Danvers, MA, USA), Bcl-2, cyclin B1, Bcl-X_L, p16, β-Actin, calreticulin thioredoxin reductase-1, and Ran-specific GTPase-activating protein (all from Epitomics, Burlingame, CA, USA). Thereafter, membranes were probed with the appropriate horseradish peroxidase-conjugated secondary antibodies (Sinbio, Shanghai, China). Bands were detected using chemiluminescence-based kit (GE Healthcare, Buckinghamshire, UK).