Primary and secondary antibodies

Total cell lysates were prepared in 1% CHAPS buffer [5mM MgCl2, 140 mMNaCl, 1mM EDTA, 1mM EGTA, 1% CHAPS, 20mM Tris-HCl (pH 7.5), and protease inhibitors (cOmplete ULTRA, Roche)]. AlgiMatrix dissolving buffer (ThermoFisher Scientific) was used to harvest spheroids before lysis in 1% CHAPS buffer. Proteins (600–1000 μg) were immunoprecipitated with BCL-2 (#4223, Cell Signaling), BCL-XL (#2762, Cell Signaling), MCL-1 (S-19, Santa Cruz) antibodies at 4°C for 16h and coimmunoprecipitates were captured by Dynabeads Protein G at 4°C for 2 h. Beads were recovered using DynaMag spin magnet and washed twice in 1% CHAPS buffer. Total cell lysates and immunoprecipitates were separated on NuPage 10% Bis-Tris gels. After SDS-PAGE, proteins were transferred onto PVDF membranes (Millipore) and then blocked with 5% dried milk in PBS-Tween20. Membranes were incubated with primary and secondary antibodies (GE Healthcare) in a buffer containing 10% milk diluent-blocking concentrate (KPL), detected with Luminata Crescendo Western HRP substrate (Millipore). Blots were imaged with LAS4000 image analyzer (Fujifilm) on chemiluminescence mode. The following antibodies were used for immunoblotting: BCL-2 (#2872, Cell Signaling), BCL-XL (#2762, Cell Signaling), Actin (#8457, Cell Signaling), BIK (#4592, Cell Signaling), MCL-1 (S-19, Santa Cruz), p53 (DO-1, Santa Cruz).

Cells were equalized (as indicated in each relevant panel), suspended in ice-cold Nonidet P-40 lysis buffer supplemented with phosphatase and protease inhibitor cocktail tablets (Roche, Mannheim, Germany). The lysate protein concentration was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). For each lane on the western blotting, an equal amount of total protein was denatured and separated with 10% SDS-PAGE gel, followed by transfer to a PVDF membrane. Both primary and secondary antibodies (GE Healthcare, Little Chalfont, UK) were applied at the dilutions recommended by the manufacturers. The primary antibodies were incubated overnight at 4 °C. The densitometry was performed using the ImageJ software (NIH, Bethesda, MD, USA). Quantifications of at least three independent experiments were shown as histograms using the software GraphPad Prism5 (La Jolla, CA, USA).

For western blotting, samples in SDS sample buffer were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes prior to blocking in TBST with 5% (w/v) milk powder or 3% (w/v) bovine serum albumin and probing with primary and secondary antibodies (GE Healthcare). Primary antibodies were: anti-Myc (Sigma, #C3956), anti-FLAG (Sigma, #F3165), anti-HUWE1 (Novus Biologicals, #NB 100-652), anti-PCF11 (Bethyl Laboratories, #A303-706A), anti-GAPDH (Cell Signaling Technology, #2118), anti-TRIM28 (Abcam, #ab22553), anti-AMPKα1 (Cell Signaling Technology, #2795), and anti-MMS19 (Proteintech, #16015-1-AP) and Tb-anti-GST (Invitrogen, #PV3551). Secondary antibodies were: Donkey Anti-Rabbit IgG (GE Healthcare, NA934V) and Sheep Anti-Mouse IgG (GE Healthcare, NA931V). FLAG and TRIM28 primary antibodies were used at a dilution of 1:10,000 and Tb-anti-GST antibody was used at a dilution 1:720 and other primary antibodies were used at a dilution of 1:1000. All GE Healthcare secondary antibodies were used at a dilution of 1:5000. Protein signal was visualized after addition of ECL detection reagent (GE Healthcare) according to manufacturer’s instructions.