Igfbp3 b 5

Preparation of total cell lysates and Western blot analysis were performed as described previously [48 (link)]. Protein content was detected using Bradford method. SDS-PAGE electrophoresis, transfer, immunostaining, and ECL detection were carried out according to standard protocols of Bio-Rad Laboratories and antibody manufacturers. Primary antibodies against the following proteins were utilized: IGFBP3 (B-5, 1:500, from Santa Cruz Biotech.), phospho-p70S6 kinase (Thr389, 1:1000), phospho-S6 (Ser240/244, 1:1000), phospho-4E-BP1 (Thr37/46, 1:1000), phospho-ERK1/2 (Thr202/Tyr204, 1:2000), ERK2 (1:3000), phospho-Akt (Thr308, 1:700), Akt (1:1000), and GAPDH (clone 14C10, 1:4000) – all from Cell Signaling Technology. Secondary antibodies for immunoblotting – GAR-HRP (1:10000) and GAM-HRP (1:10000) were also from Cell Signaling Technology.

Cells were lysed using Mammalian Protein Extraction Reagent (M-PER) (Thermo Fisher Scientific) containing protease inhibitor cocktail (cOmplete tablet; Roche, Mannheim, Germany) and phosphatase inhibitor cocktail (PhosSTOP; Roche). Equal amounts of proteins from cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked using 5% ECL blocking agent (GE Healthcare, Chicago, IL) in Tris-buffered saline containing 0.05% Triton X-100. Proteins of interest were detected using primary antibodies against phosphorylated forms of AKT (Ser473; Cell Signaling Technology, Danvers, MA) and ERK1/2 (PROMEGA, Madison, WI), and against IGFBP-3 (B-5; Santa Cruz Biotechnology, Dallas, TX), AKT (Cell Signaling) and β-actin (clone C4; Millipore, Billerica, MA). Following incubation with primary antibodies, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Detected proteins were visualized with ECL Western Blotting Detection reagents (GE Healthcare). Protein expression levels were quantified using ImageJ^{56 (link)}. Active MAPK/ERK and p-AKT expression levels were normalized to β-actin and total AKT protein levels, respectively.