Western Blotting of Phosphorylated Proteins

Differentially treated cells were harvested, and 3 to 10 µg of protein per sample was used for Western blotting as described before [63 (link),97 (link)]. Used primary antibodies were anti-phospho-GSK3b (1:250, rabbit; #9336, Cell Signaling, Danvers, MA, USA), anti-phospho-Akt (1:250, rabbit; #4060, Cell Signaling), anti-phospho-mTOR (1:250, rabbit; #2971, Cell Signaling), anti-phospho-4E-BP1 (1:1000, rabbit; #2855, Cell Signaling), and anti-phospho-P70S6K (1:1000, rabbit; #9205, Cell Signaling) in 5% bovine serum albumin/tris-buffered saline with 0.1% Tween 20 (TBST). Secondary antibody was donkey-anti-rabbit IgG-HRP (1:12,500; A16035, Thermo Fisher Scientific) in 2% (w/v) casein/TBST. Loading of equal amounts of protein was confirmed by stripping and incubation of the membranes with anti-GAPDH (1:200, mouse; sc-47724, Santa Cruz Biotechnology, Dallas, TX, USA) in 2% (w/v) casein/TBST and the secondary antibody donkey-anti-mouse IgG-HRP (1:10,000; A16011, Thermo Fisher Scientific) in 2% (w/v) casein/TBST. The signal densities were quantified using ImageJ^® software. The signals were normalized to GAPDH, and the n-fold signal induction was determined in relation to the respective control samples (control = 1).