Anti mcl 1

Mitochondria and Nucleus fractions were obtained using the Mitochondria Isolation Kit (Product No. 89874) and NE-PER™ Nuclear and Cytoplasmic Extraction kit (Product No.78833) for cultured cells and tumor samples from Thermo Fisher Scientific, Waltham, MA according to manufacturer’s instruction. Briefly, after 24–48 h doxorubicin (100 ng/ml) or oxaliplatin (10 μM) treatment, 2 × 10⁷ HCT116 p53^−/− cells, Colo205, SW480, HT29, CRISPR ENO1 cells or tumor cells were harvested by centrifuging at ~850 × g for 2 min, and mitochondria or Nucleus were isolated following the protocol provided by the kit. Mitochondria pellets and nucleus lysates were lysed in RIPA buffer. After removing the insoluble material by 14,000 × g centrifugation, protein from mitochondria or nucleus was quantified by BCA Assay kit (Product No. 23225, Thermo Fisher Scientific, Waltham, MA). For gel electrophoresis, 30 μg of total protein was loaded per lane and separated by SDS-PAGE, and then transferred to PVDF membrane (Bio-Rad, Hercules, CA). For immunoblot experiments, the membranes were sequentially blotted with, anti-Tom20 (Santa Cruz Biotechnology, Dallas, TX), anti-MCL1 (Cell Signaling Technology, Danver, MA) and Histone H3 (Santa Cruz Biotechnology, Dallas, TX) primary antibodies, and horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA).

Western blotting was performed as described previously [62 (link)]. The primary antibodies used in this study include anti-γH2A.X (1:1000, 2577S, Cell Signal Technology, Danvers, MA, USA), anti-p53 (1:500, sc-6243, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p53ser15 (1:1000, 9286S, Cell Signal Technology, Danvers, MA, USA), anti-cleaved PARP1 (1:1000, 5625S, Cell Signal Technology, Danvers, MA, USA), anti-MCL1 (1:1000, 39224S, Cell Signal Technology, Danvers, MA, USA), anti-BCL2 (1:1000, 2872S, Cell Signal Technology, Danvers, MA, USA), anti-ACTIN (1:150,000, MAB1501, Millipore, Burlington, MA, USA). The secondary antibodies used were HRP-linked anti-rabbit IgG (1:4000, 7074S, Cell Signaling Technology, Danvers, MA, USA) and HRP-linked anti-mouse IgG (1:4000, 7076S, Cell Signaling Technology, Danvers, MA, USA).

Western blot was performed as previously described [19 (link)]. Primary antibodies used were anti-BIM (1:1000, #2933 (C34C5) Cell Signaling Technology, Danvers, MA, USA), anti-MCL-1 (1:1000, #5453 (D35A5) Cell Signaling Technology), anti-BCL-2 (1:1000, ab32124 (E17), Abcam, Cambridge, United Kingdom), and anti-BCL-XL (1:1000, #2764 (54H6) Cell Signaling Technology).

Cells growing in Petri dishes were collected and lysed in cell lysis buffer (Cell Signaling Technology, Boston MA USA) containing 20 mmol/L Tris-HCL (PH7.5), 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGDA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO4, 1 μg/ml leupeptin, and 1 mmol/l phenylmethane sulphonyl fluoride. Membranes were incubated overnight at 4°C with specific antibodies in TBS-T (TBS-0.1% Tween20) and subsequently for one hour with the appropriate horseradish-peroxidase-conjugated anti-rabbit/mouse secondary antibody. The following mouse monoclonal antibodies were used: Anti-CyclinB1 (Santa Cruz Biotechnology, Dallas Texas USA), anti-Caspase-8, anti-β-actin (both from Cell Signaling Technology, Boston MA USA). The following rabbit polyclonal antibodies were used: anti-Caspase-3, anti-PARP, anti-Bid, anti-Bad, anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Mcl-1 (all from Cell Signaling Technology), anti-c-MET, anti-phosphorylated c-MET (Tyr1349 and Tyr1234/1235). For Caspase-3/7 activity assays the Apo-ONE® Reagent kit (Promega, Madison WI USA) was used according to the instructions of the manufacturer. Actin was used for control of appropriate protein load for each membrane. In figures, one actin loading control has been exemplarily shown.

Whole cell lysates were prepared in NP-40 lysis buffer57 (link). Immunoblotting was performed with anti-MUC1-C58 (link), anti-MCL-1, anti-phospho-ERK, anti-ERK, anti-phospho-AKT(Ser-473), AKT, anti-phospho-GSK3β(Ser9), anti-GSK3β, anti-phospho-MCL-1(Thr163) (Cell Signaling Technology Inc), anti-phospho-MCL-1(Ser-159) (Abcam) and anti-β-actin (Sigma-Aldrich Co).

All the cells were harvested and lysed with ice-cold RIPA buffer (Merck Millipore, Burlington, MA, USA), protease inhibitor cocktail (Sigma-Aldrich), and phosphatase inhibitor cocktail (Sigma-Aldrich). Equal amounts (30 μg) of protein were resolved through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore). After blocking with 5% skim milk for 1 h, the PVDF membranes were incubated with primary antibodies, such as anti-VEGF 165A (Abcam PLC, Cambridge, England, UK), anti-ROCK1 (Abcam PLC, Cambridge, England, UK), anti-Met (Cell Signaling Technology, Danvers, MA, USA), anti-HIF-1α (Cell Signaling Technology), anti-Mcl-1 (Cell Signaling Technology), and anti-β-Actin (Sigma-Aldrich), overnight at 4° C. Following washing with tris-buffered saline containing Tween 20 (TBS-Tween 20), the PVDF membranes were incubated with the secondary antibody at room temperature for 1 h. After the membranes were washed, immunoreactive proteins were detected using a SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).