Phospho p70s6kinase

LPA-induced signaling was further examined by determining the ratio of the phosphorylated form of each protein to the total amount of the protein using Western analysis. Whole cell lysates were separated by 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to a PVDF membrane using a transfer apparatus according to manufacturer’s protocol (Bio-Rad, Hercules CA). After incubation with 5% bovine serum albumin (BSA, fraction V; Sigma) in PBST (10 mM sodium phosphate, pH 8, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was rinsed once with PBST and incubated with antibodies against phosphorylated CREB (S133) (1:1000, #9191), phospho-Erk1/2 (Erk1/2; Thr202/Tyr204; 1:1000, #9101) and Erk1/2 (1:1,000, #9102), phospho-p70S6kinase (T389) (1:1000, #9205 and #9234), p70 S6 kinase (T389) (1:1000, #9205 and #9234) (all from Cell Signaling Technology, Danvers MA), phospho-p70 S6 kinase (1:1000, MAB8963, R&D Systems), GAPDH (1:20000, sc47724, Santa Cruz Biotechnology, Dallas, TX) at 4 °C overnight. Membranes were washed three times for 10 min and incubated with 1:5000 dilution of anti-rabbit or anti-mouse secondary antibodies for 1 h. The membrane was washed three times for 15 min with PBS and detected using a chemiluminscent detection system (ECL; Thermo Fisher) using manufacturer’s protocol.