Igf irβ

Protein isolation and western blots were performed as previously described [119 (link)]. Primary antibodies were used against IGF-IRβ (1:1000), phospho-IGF-IR/IR (1:500), phospho-AKT (1:1000), pan-AKT (1:1000), AKT1 (1:1000), AKT2 (1:500) phospho-ERK1/2 (1:500), ERK (1:1000) and β-actin (1:5000) (Cell Signaling Technology, Danvers, MA) and against human (transgenic) IGF-IR (1:1000) (R&D Systems, Minneapolis MN) overnight at 4°C. Membranes were then incubated with either an anti-rabbit (1:2000) (Cell Signaling Technology, Danvers, MA) or anti-goat (1:2000) (Santa Cruz Biotechnology. Dallas, TX) secondary antibody for one hour at room temperature. Membranes were visualized using chemiluminescence substrate (PerkinElmer, Waltham, MA) and FluoroChem 8800 gel documentation system (Alpha Innotech - ProteinSimple, Toronto, ON).

Following treatment with BMS-745807, adherent cells were lysed, protein was isolated and western blots performed as previously described [69 (link)]. Primary antibodies were used against IGF-IRβ (1:1000; cat# 3027), phospho-IGF-IR (Tyr1135/1136)/IR (Tyr1150/1151) (1:500; cat# 3025), phospho-Akt(Ser473) (1:1000; cat# 4060), phospho-ERK1/2 (Thr202/Tyr204) (1:500; cat# 4377), and β-actin (1:5000; cat# 4970) (Cell Signaling Technology, Danvers, MA) overnight at 4 °C. An anti-rabbit secondary antibody (1:2000; cat# 7074) (Cell Signaling Technology, Danvers, MA) was used for 1 h at room temperature. Membranes were visualized using chemiluminescence substrate (PerkinElmer, Inc., Waltham, MA) and a FluorChem 8800 gel documentation system (Alpha Innotech—ProteinSimple, Toronto, ON) or a ChemiDoc XRS + imaging system (Bio-Rad Laboratories, Mississauga, ON). For western blots captured using the FluorChem 8800 gel documentation, densitometry of the bands was determined using AlpahEase FC software (Alpha Innotech—ProteinSimple, Toronto, ON) while western blots captured using the ChemiDoc XRS + gel documentation, densitometry of the bands was determined using ImageLab 5.2.1 software (Bio-Rad Laboratories, Mississauga, ON).