Bcl xl

Cells seeded and cultured for 24 h were lysed in RIPA lysis buffer and protein concentrations determined as above. Colorectal and melanoma PDX tumour tissue was homogenised and protein concentrations determined as described above. Recombinant protein standards for MCL1 (Cloud-Clone Corp, Texas, USA) and BCL-X_L (R&D Systems, Abingdon, Oxfordshire, UK) were diluted in RIPA with 1 × Laemmli sample buffer to a concentration range of 2.5–100 nM. SDS-PAGE was then performed with 5–6 standards of 0.0375 pmol to 1.5 pmol MCL1 or BCL-X_L protein to determine the levels of MCL1 and BCL-X_L in 15 to 20 µg of total cellular protein by western blotting with anti-MCL1 antibody (Santa Cruz Biotechnology, Dallas, USA; sc-819) and anti-BCL-X_L antibody (Cell Signalling Technology, NEB, Hitchin, UK; 2762) and using anti-rabbit fluorescently labelled secondary antibodies (Cell Signalling Technology, NEB, Hitchin, UK) diluted 1:15,000 and infrared imaging for quantification (LI-COR, Cambridge, UK).

Cells were lysed in the laemmli buffer (Biorad) containing 1X protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA, 78440). Cell lysates were run on a 4–12% SDS PAGE gel (Invitrogen NP0321BOX), the proteins were transferred to a PVDF membrane, and the membrane was blocked with 5% skim milk (VWR (Bridgeport, NJ, USA) 97063-958) in TBST (0.1% Tween20) and probed with target antibodies. Primary antibody incubations were performed overnight at 4 °C. Antibodies were used Rpb1 (CST 14958), p-Rpb1 (CST 4735), PARP (CST 9532; 1:500), cCP9 (CST 7237; 1:500), cCP3 (CST 9665; 1:500), Mcl-1 (CST 5453; 1:500), Bcl-xL (CST 2764; 1:500), Bcl-2 (CST 4223; 1:500), Noxa (Calbiochem (Burlington, MA, USA) OP180, clone 114C307; 1:500), β-actin (Sigma Aldrich A1978, clone AC15; 1:2000). The HRP linked secondary antibodies were from Santa Cruz Biotechnology Inc. The target protein was detected on the Azure (C300) imaging system. In selected cases, the protein capillary electrophoresis (Protein Simple (San Jose, CA, USA) SM-W004) was used. The following antibodies were applied Mcl-1 (CST 5453; 1:25), Bcl-xL (CST 2764; 1:25), Bcl-2 (R&D System, MN, USA, MAB827; 1:25), Noxa (Calbiochem OP180, clone 114C307; 1:25), and Vinculin (Abcam (Cambridge, MA, USA) ab129002, 1:500).

The antibodies used are as follows: caspase-3 (9661 and 9662, Cell Signaling, Beverly, MA, USA), Bcl-2 (ab7973, Abcam, Cambridge, MA, USA), Mcl-1 (ab53709, Abcam), Bcl-xL (AF800, R&D Systems, Minneapolis, MN, USA), cytochrome c (556433, BD Biosciences, San Diego, CA, USA), Bax (2772, Cell Signaling; 2280, Trevingen), L-aromatic amino acid decarboxylase (AV41425, Sigma), Thiophosphate ester (ab92570, Abcam), Cdk2 (ab7954, Abcam, polyclonal rabbit), cytochrome c Oxidase (459600, Invitrogen, Carlsbad, CA, USA) and Caspase-7 (9492, Cell Signaling)