Phosph mek1 2 ser 217 221

Tissues and cultured cells were homogenized in lysis buffer supplemented with protease and phosphatase inhibitors (Thermo Scientific), and 50 μg of total protein was resolved by SDS-polyacrylamide electrophoresis under reducing conditions. Proteins transferred to nitrocellulose membranes were probed with antibodies: phosphop53 (Ser15) (1:1000; Cell Signaling), p53 (1:2000; (CM5) Leica Microsystems and 1:1000; (1C12) Cell signaling), PARP (1:1000; Cell Signaling), phospho-p70 S6 Kinase (Thr 389) (1:1000; (108D2) Cell Signaling), p70 S6 Kinase (1:500; (C19) Santa Cruz Biotechnology), p70 S6 Kinase (1:2000; (H-9) Santa Cruz Biotechnology), phospho-AKT (Ser 473) (1:2000; (193H12) Cell Signaling), AKT (1:2000; Cell Signaling), phosph-MEK1/2 (Ser 217/221) (1:2000; Cell Signaling), MEK-1 (1:2000; (C-18) Santa Cruz Biotechnology), phospho-ERK (1:2000; (E-4) Santa Cruz Biotechnology), ERK1 (1:2000; (K-23) Santa Cruz Biotechnology), tubulin (1:4000; (DM 1A) Sigma), actin (1:2000; (20 (link)-33 ) Sigma) and GAPDH (1:10,000; (D16H11) Cell Signaling). Detection was performed with horseradish peroxidase–conjugated secondary antibodies and ECL Plus Western blotting Detection System (GE Healthcare). Data quantification was determined by relative densitometric values of scanned radiographic films.