P gsk3beta ser9

The cells were lysed by boiling in SDS‐loading buffer containing 0.1 M Tris (pH 6.8), 16% v/v glycerol, 3.2% w/v SDS, 10% v/v β‐mercaptoethanol and 0.005% w/v bromophenol blue. Sample loading was normalized according to protein concentration determined by DC protein assay (Bio‐Rad). Cell lysates were subjected to SDS‐PAGE and electroblotted to PVDF membrane. The blots were probed with the pAkt (Ser473)‐, Akt‐, p‐p70S6K (Thr421/Ser424)‐ and pGSK3beta (Ser9)‐specific antibodies (Cell Signaling Technology, Beverly, MA, USA) or the anti‐alpha‐tubulin antibody (Sigma‐Aldrich) according to instructions of manufacturers. The blots were developed using secondary antibodies conjugated to peroxidase (Cell Signaling Technology; Sigma‐Aldrich) and standard ECL using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).

Treated and control whole cell lysates were prepared in lysis buffer (Cell Signaling, Danvers, MA, USA). Eighty to one-hundred μg of protein were electrophoresed in a 10% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Proteins were electro-blotted onto PVDF membrane in a 50 mM Tris-base, 20% methanol, and 40 mM glycine electrophoresis buffer. Membranes were incubated in 5% non-fat dry milk in PBST (Phosphate 100 mM, KCl 27 mM, NaCl 1.37 M pH 7.4 after 1X dilution; 0.2% Tween-20) for 1 h. Blots were probed with primary antibody overnight at 4°C in 2% BSA in PBST, and then incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling) in 5% dry milk in PBST for 1 h at room temperature. Bound antibodies were detected by Super Signal West Pico Chemiluminescent Substrate detection reagent (Pierce/Thermo Scientific, Rockford, IL, USA) and visualized by autoradiography. The primary antibodies used for Western blot analysis were: anti-GLIPR1 (Novus Biologicals, Littleton, CO, USA), p-AKT Ser473 (Cell Signaling), p-GSK3beta Ser9 (Cell Signaling), p-PDK1 Ser241 (Cell Signaling), anti-CX3R1, GAPDH and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA).