Western Blot Analysis of Signaling

Cell lysates were prepared and subjected to western blot (WB) analysis as described in detail elsewhere [35 (link)]. Briefly, cells were lysed in LDS sample buffer and analyzed by SDS-PAGE on 4-12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA). Following electrophoresis, proteins were transferred to nitrocellulose membrane, blocked, and incubated overnight with appropriate primary antibody. Following washing, they were incubated with HRP-labeled secondary antibody from Pierce (Rockford, IL, USA). Detection was performed using enhanced chemiluminescent substrate (Pierce) and exposure to X-ray film or imaged using the Odyssey system (Li-cor, Lincoln, NE, USA). Primary antibodies diluted in 5% BSA in TBST for IGF-1R (1:2,000), phospho-ERK1/2 (1:2,000), total ERK1/2 (1:2,000) phospho-Akt (1:2,000) and phospho-IGF-1R (1:2,000) were from Cell Signaling Technologies (via BioNordika, Stockholm, Sweden). Primary antibodies diluted in 5% non-fat dry milk in TBST for p53 (sc-126, 1:1,000) and GAPDH (sc-25778, 1:4,000) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).