Rabbit monoclonal anti phospho akt ser473

Cells were lysed with SDS sample buffer (70 mM Tris-HCl, 3% SDS, and 10% glycerol) with inhibitor cocktails of protease and phosphatase. Primary antibodies were rabbit monoclonal anti-phospho-p44/42 kinase (Thr202/Tyr204: Cell Signaling Technology, Danvers, MA, USA), anti-p44/42 kinase (Cell Signaling Technology), rabbit monoclonal anti-phospho-Akt (Ser473:Cell Signaling Technology), rabbit monoclonal anti-Akt (Cell Signaling Technology), rabbit polyclonal anti-phospho-p70 S6 kinase (Thr389: Cell Signaling Technology), rabbit polyclonal anti-p70 S6 kinase (Cell Signaling Technology), and mouse monoclonal anti-β-actin-peroxidase (Sigma-Aldrich, St. Louis, MO, USA). For stimulation experiments, cells were starved for 24 h with serum-free DMEM and then stimulated with 100 ng/ml of CCL5. Cell lysates were obtained after the indicated times stimulation and subjected to SDS-polyacrylamide gel electrophoresis, immunoblotted with the primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies, and then analyzed by a lumino image analyzer.

Cell extracts were resolved in a 10% SDS-PAGE gel and transferred to nitrocellulose filter membrane. The antigens were detected by specific antibodies followed by the respective secondary antibodies (LI-COR Biotechnology, Lincoln, NE, USA). GAPDH was simultaneously detected as a loading control. Detection was performed with an Odyssey infrared imaging system (LI-COR Biotechnology). The following antibodies were used: rabbit monoclonal anti-SHP2, rabbit monoclonal anti-STAT3, and rabbit monoclonal anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit monoclonal anti-ERK, rabbit monoclonal anti-AKT, rabbit monoclonal anti-phospho-ERK, rabbit monoclonal anti-phospho-STAT3 (Tyr705) and rabbit monoclonal anti-phospho-AKT (Ser473) (Cell Signaling Technologies, Beverly, MA, USA).