Anti phopho s6 ser 235 236

The following antibodies were purchased from Cell Signaling Technology anti-Pim1 (Cat#2907), anti-S6(Cat#9202), anti-phopho-S6 (Ser 235/236 Cat#4856), anti-EDC3 (Cat #14495), anti-phospho-GSK3B, (Ser9, Cat#9336), anti-phopho-IRS1 S1101 (Cat#2385), anti-phospho-eIf4B S406 (Cat #5399), anti-eIF4B (Cat#3592), anti-AKT (Cat#9272), and anti-phopho-AKT S473 (Cat #4058), anti- phosphor 4E-BP1 (T37/46) (Cat#2855), anti-4E-BP1 (Cat#9644), c-Myc (Cat# 9402). The anti-phospho-EDC3 (S161 Cat#600-401-J38) was purchased from Rockland Antibodies while the HRP conjugated anti-β-actin (Cat#A3854) was from Sigma. Anti-IRS1 (Cat #06-248) was obtained from Merck Millipore. Antibodies bought from Santa Cruz Biotechnology included anti-EDC3 (Cat# sc-365024) and anti-GAPDH HRP (Cat. #0411). The kinase inhibitors BKM 120 (Cat# A11016) and AZD5363 (Cat#A11759) were purchased from Adooq Bioscience. VBT5445 was purchased from Chembridge.

Cells were lysed in RIPA buffer (Pierce) supplemented with protease and phosphatase inhibitor cocktails (Thermo Scientific). Blots were incubated with anti-MCL1 (1:1000, Cell Signaling), anti-phopho-MCL1 Ser159 (1:1000, Abcam), anti-phopho-MCL1 Thr163 (1:1000, Cell Signaling), anti-cleaved caspase 3 (CC3) (1:250, Cell Signaling), anti-β-actin (1:10000, Abcam), anti-vinculin (1:20000, Sigma-Aldrich), anti-BCL2 (1:500, Cell Signaling), anti-BCLXL (1:1000, Cell Signaling), anti-BIM (1:1000, Cell Signaling), anti-phospho-AKT Thr308 (1:1000, Cell Signaling), anti-phospho-AKT Ser473 (1:1000, Cell Signaling), anti-phopho-S6 Ser235/236 (1:3000, Cell Signaling), anti-phopho-ERK1/2 Thr202/Tyr204 (1:1000, Cell Signaling), anti-p21 (1:1000, Santa Cruz Biotechnology), anti-p27 (1:1000, Cell Signaling), anti-p53 (1:1000, Santa Cruz Biotechnology), anti-c-MYC (1:1000, Santa Cruz Biotechnology), anti-β-catenin (1:1000, Cell Signaling), anti-phospho-EGFR Tyr845 (1:500, Cell Signaling), anti-phospho-EGFR Tyr1068 (1:500, Cell Signaling), anti-EGFR (1:1000, Cell Signaling), anti-HA (1:1000, Cell Signaling), anti-TRIM21 (1:1000, Bethyl Laboratories), or anti-USP9X (1:1000, Bethyl Laboratories), and then with 1:5000 of anti-rabbit or anti-mouse secondary antibodies (Promega).