Phospho akt ser473 clone d9e

Cells were lysed in cold RIPA lysis buffer and Halt™ Protease and Phosphatase Inhibitor cocktail (Thermo Scientific, Waltham, MA, USA) for 20 min on ice. The cell lysates were clarified by centrifugation at 10,000×g for 20 min. Proteins (10–25 μg) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked in TBS-T buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) containing 5% (w/v) non-fat milk at room temperature for 1 h and then probed at 4 °C overnight with antibodies to detect AKT (pan) (clone C67E7, 1:1000), phospho-AKT (Ser473) (clone D9E, 1:1000), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (clone D13.14.4E, 1:1000), p44/42 MAPK (ERK1/2) (clone 137F5, 1:2000) from Cell Signaling Technology (Boston, MA, USA); Gαq (clone ab75825 and ab190082, 1:1000) from Abcam (Cambridge, USA); DYKDDDDK Tag Monoclonal Antibody (FG4R, MA1-91878, 1:1000) from Thermo Scientific (Waltham, MA, USA), and GAPDH (clone 60004-1-Ig, 1:2000) from ProteinTech (Chicago, IL, USA). Detection was carried out with the SuperSignal West Femto Maximum Sensitivity Substrate Trial kit (Pierce, Rockford, IL, USA). The band images were digitally captured and quantified with a ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Hercules, CA, USA).