Anti mcl 1 cell signaling

Cells were lysed in NP40 lysis buffer (0.5% NP40, 50 mM Tris pH 8, 120 mM NaCl, with the addition of PhosSTOP phosphatase inhibitor tablets (Roche, #4906845001) and cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche, #11836170001). 250 μg of total lysate was incubated with 2.5 μl Bim antibody (Cell Signaling, #2819), 1 μg Mcl-1 antibody (Santa Cruz, #sc-12756), normal rabbit IgG (Millipore, #12-370) or normal mouse IgG (Millipore, #12-371) overnight at 4°C. The following day 50 μl of Protein A (Pierce, #88846) or Protein G (Pierce, #88847) magnetic beads were incubated with lysates for 1 hour for Bim IP and Mcl-1 IP, respectively. Beads were washed 3 times with lysis buffer and incubated in 2x sample buffer for 15 min at room temperature. Eluted proteins were separated from the beads and boiled for 10 min. For Bim IP, anti-USP9X Cell Signaling #14898, anti-Mcl-1 Cell Signaling #5453 and anti-Bim Cell Signaling #2819 antibodies were used for western blot detection. For Mcl-1 IP, anti-Mcl-1 Abcam #ab32087 and anti-Bim Cell Signaling #2819 antibodies were used for western blot detection.

PANC 10.05 and CAPAN2 cells were transfected with pRK5-HA-Ubiquitin-WT (gift from Ted Dawson, RRID: Addgene_17608). 24 hours after transfection, cells were treated with trametinib or DMSO (control) +/- 10 μM MG132 2 h prior to lysis. Cells were lysed in RIPA buffer (0.1% SDS, 1.0% Triton-X 100, 150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris pH 8.0) containing PhosSTOP phosphatase inhibitor tablets (Roche, #4906845001), cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche, #11836170001) and 100 mM N-Ethylmaleimide (NEM). 1 mg of total protein was incubated with anti-HA magnetic beads (Pierce, #88836) for 2 h. Beads were washed 3 times with lysis buffer and incubated in 2x sample buffer for 15 min at room temperature. Eluted proteins were separated from the beads and boiled for 10 min. Anti-Mcl-1 Cell Signaling #94296 was used for immunoblots.