Anti p akt ser129

Total protein (50 µg) extract of SNU-1 cells was prepared with cell lysis buffer (50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM, Sodium orthovanadate, 1 mM NaF, 10 lg/ml aprotinin, 10 lg/ml, leupeptin, 1 mM PMSF). Then, 20 μg of proteins was separated on SDS polyacrylamide gels and transferred to PVDF membranes (Immobilon-P transfer membrane, Bedford, MA, USA). The primary antibodies used were the following proteins: p-CK2 (pS/ pTDXE, #8738), AKT (Ser473, #9272), mammalian target of rapamycin (mTOR) (7C10, #2983), p-mTOR (Ser2448, #2971), p70S6K (#9202), p-p70S6K (Thr387, #9234), cleaved poly(ADP-ribose) polymerase (PARP) (Asp214, #9541) (all from Cell Signaling Technology, Danvers, MA, USA); anti-p-AKT (Ser129, ab133458, Abcam, Cambridge, UK); and CK2 (1AD9, sc-12738, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing, the blots were incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as secondary antibodies and visualized using super ECL detection (Amersham Pharmacia Biotech, Buckinghamshire, UK).