Anti total s6 ribosomal protein

Cultured cells in log-phase growth were treated with 100 nM of MEK162 for 10 min, 24 and 48 h. The plates were then washed twice with ice-cold PBS, lysed and collected using mild lysis buffer. Lysates were centrifuged at 10 000 r.p.m. at 4 °C for 10 min to clear insoluble material, and the resulting supernatant was collected and quantified using a bicinchoninic acid assay (Pierce Biochemicals, Rockford, IL, USA). Protein was resolved by SDS–PAGE and transferred to nitrocellulose membranes (Invitrogen). Anti-phospho-AKT (Thr308 cat # 9275), anti-total AKT (cat # 92725), anti-phospho-ERK1/2 (T202/Y204 cat # 9101), anti-total ERK1/2 (cat # 9102), anti-phospho-S6 ribosomal protein (Ser235/236 cat # 2211), anti-total S6 Ribosomal Protein (SG10 cat # 2217), anti-phospho-RB (Ser780 cat # 9307), anti-total RB (cat # 9309), anti-cyclin D1(cat # 2921), anti-phospho cyclin D1 (cat # 2922), anti-phospho-FoxO1(Thr24)/FoxO3a (Thr32) (cat # 9464), anti-p27kip (cat # 2552), anti-beta-actin (cat # 4967), and anti-alpha-tubulin (cat # 2144) antibodies were obtained from Cell Signaling Technologies (Danvers, MA, USA).