Quantifying Protein Expression by Western Blot

The detection of protein levels was performed by Western blotting as described [10 (link), 50 (link), 51 (link)]. In brief, extracted protein concentration was determined using the BCA protein assay (Sigma, St. Louis, Mo). For electrophoresis, 20 µg of total protein was separated on Criterion TGX Stain-Free gels (4–20%, Bio-Rad). Stain-free gels were then imaged prior to transfer on a Bio-Rad Chemidoc MP Imaging System (Bio-Rad, Hercules, CA) to confirm equal protein loading. Gels were then wet transferred onto PVDF membranes at 100 V constant for 1 h, and equal transfer was confirmed by Ponceau S. Membranes were then blocked in 5% milk in TBST for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies from cell signaling against p-Akt^Thr308 (1:1000; #13,038), total Akt (1:1000; #4691), p-p44/42MAPK^{Thr202/Tyr204} (1:1000; #9101) total p44/42 MAPK (1:1000; #4695), total IGF-1R (1:1000; #9750), and InsRβ (1:1000; #3025). Following a 1-h incubation with the appropriate secondary antibody. Clarity Western ECL Substrate (Bio-Rad, Hercules, CA) was applied to the membrane and bands were visualized using a Bio-Rad Chemidoc MP bioimager to first pixel saturation and densitometry was performed using Image Lab software (Bio-Rad, Hercules, CA).