Rabbit monoclonal anti phosphorylated akt ser473

Protein extracts were prepared from tissue samples (∼50 mg) by homogenization in 500 μl whole cell lysis buffer (1% NP-40, 10 mM Tris pH 7.8, 150 mM NaCl, 40 mM EDTA, 10 mM Na-Pyrophosphate, 10 mM NaF, 1mM PMSF, 4 mM Orthovanadate, Minicomplete iprotease inhibitor^®). Protein extracts (30 μg) were separated by polyacrylamide gel electrophoresis. Blottings were incubated one hour at RT in a blocking buffer containing 5% skim milk and then incubated overnight at 4°C with mouse monoclonal anti-phosphorylated STAT3 (Santa Cruz), rabbit monoclonal anti phosphorylated AKT (Ser473) (Cell Signaling, Danvers, MA), mouse monoclonal anti Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), mouse monoclonal anti Diphosphorylated-JNK (JNK-PT48) (Sigma) and beta-actin mouse monoclonal antibody (Sigma-Aldrich), and subsequently, with peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (Dako) for one hour at room temperature.

Cells grown and treated as indicated were collected, and the total protein was extracted. The following primary antibodies were used: rabbit monoclonal anti-phosphorylated IGF-1R (Tyr1131), rabbit monoclonal anti-IGF-1R, rabbit monoclonal anti-Akt, rabbit monoclonal anti-phosphorylated Akt (Ser473), rabbit monoclonal anti-phosphorylated mTOR (Ser2448), rabbit monoclonal anti-mTOR, rabbit monoclonal anti-p70s6k, or rabbit monoclonal anti-phosphorylated p70s6k (Thr389), rabbit monoclonal anti-S6, or rabbit monoclonal anti-phosphorylated S6 (Ser240/244), rabbit monoclonal anti-AMPK, or rabbit monoclonal anti-phosphorylated AMPK (Thr172) (all from Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat-anti-rabbit antibody (Thermo Scientific) was used as a secondary antibody. The control for equal protein loading was assessed with an anti-β-actin antibody (Cell Signaling Technology, Inc.).