Anti mcl 1

Z-VAD was purchased from Calbiochem. The antibodies used were as follows: anti-caspase-3 (Cell signaling, 9662), anti-caspase-8 (Cell signaling, 9746), anti-caspase-8 (Santa Cruz Biotech, SC-6136), anti-caspase-9 (Cell signaling, 9508), anti-TRADD (Cell signaling, 3684), anti-actin (Sigma, A2066), anti-Flag (Sigma, F-3165), anti-tubulin (Sigma, T7816), anti-PARP (Cell signaling, 9542), anti-Mcl1 (BD, 554103), anti-TL1A (Enzo, ALX-804-859-C100), and anti-TL1A (PEPROTECH, 500-P240). Recombinant proteins: FasL (R&D, 126-FL/CF), TNFα^{24 (link)} and TRAIL^{25 (link)}. ELISA kits: TL1A (ENZO Life Science, APO-54N-027), TNF-alpha (RayBiotech, ELH-TNFα), FasL (RayBiotech, ELH-FASL), and TRAIL (RayBiotech, ELH-TRAIL).

Protein expression was determined by Western blot analysis, as previously described [12 (link)]. The following antibodies were used: anti-Mcl-1 (1:1000; #559027, BD Pharmingen, Franklin Lakes, NJ, USA), anti-Bcl-2 (1:1000; #551098, BD Pharmingen, Franklin Lakes, NJ, USA), anti-Bad (1:1000; #9292, Cell Signalling, Frankfurt, Germany), anti-Bax (1:1000; #2772, Cell Signalling, Frankfurt, Germany), anti-Bcl-xL (1:1000; #610211, BD Pharmingen, Franklin Lakes, NJ, USA), anti-Bim (1:1000, #2819, Cell Signalling, Frankfurt, Germany), anti-β-actin (1:10000; #A5441, Sigma-Aldrich, St. Louis, MO, USA). Secondary HRP-linked antibodies were purchased from Jackson ImmunoResarch (#115-035-006, #111-035-006, West Grove, PA, USA).

Cells were lysed either in 8 M Urea or in RIPA buffer (Thermo Fisher, #89900) and boiled in Laemmli-buffer containing DTT. Samples were heated for 5 min. Antibodies used were: Bak(NT), Bak(aa23-38), Bak(Ab-1), Bak(G23) as above. The following antibodies were purchased from Cell Signaling unless indicated otherwise: anti-Bim (# C34C5), anti-GAPDH (Millipore, #MAB374), anti-Bcl-X_L (#54H6), anti-Bcl-2 (#2870), anti-Bax (#2772), anti-VDAC (#4661), anti-Hsp60 (#4870), anti-Mcl-1 (BD, #559027), anti-Hsp60(Ctr) (Enzo Life Sciences, #ALX-804-072), anti-Tom22 (Santa Ccruz, sc-58308), anti-Bcl-w (#2724 S), anti-GFP (Roche, #11814460001), anti-cytochrome c (#11940) and anti-tBid [64 (link)], anti-BiP (Cell signalling, #3177), anti-VDAC2/3 (Thermo Fisher, #PA141205). Peroxidase-conjugated secondary antibodies were goat anti-rabbit IgG (Sigma, #A6667), goat anti-rabbit Fc (Sigma, #AP156P), goat anti-mouse IgG (Dianova, #115035166), goat anti-mouse Fc (Sigma, #AP127P) and goat anti-rat IgG (Dianova, #112035062).

Cells were lysed using ice cold CHAPS lysis buffer (1% CHAPS, 20 mM TRIS-HCL pH 7.5, 137 mM NaCl, 5 mM MgCl₂, 1 mM EDTA and 1 mM EGTA), protease inhibitor cocktail (P8340), phosphatase inhibitor cocktail 2 (#P5726) and 3 (#P0044) (Sigma) according to manufacturer’s instructions. For immunoprecipitation, lysates were incubated overnight at 4 °C with Protein G Dynabeads™ (Invitrogen) and the following antibodies: anti-MCL-1 (12 µg; BD Pharmingen), anti-BCL-xL (8 µg; EMD Millipore), and anti-mouse IgG1 (12 µg; #5415S) and IgG3 (8 µg; #37988 S) isotype control (Cell Signaling Technology). The immunoprecipitate (IP) was divided equally to blot for MCL-1, BCL-xL, and BIM. Twenty micrograms of the supernatant, and the initial whole-cell lysate for each condition (input) was loaded onto the gel with the IP and analyzed by Western blot analysis (as previously described).

Frozen cell pellets were lysed in 50 mM Tris–HCl, pH 7.5, 10% glycerol, 5 mM MgCl₂, 1 mM EDTA, and 0.5% CHAPS. Protein content of lysates was determined by Pierce 660 nm protein assay reagent (ThermoFisher Scientific) and used to normalize gel loading. Lysates were run on NuPAGE gels (Invitrogen) using either MOPS or MES (Fig. 4e and Fig. S3d) running buffer before being transferred to a PDVF membrane, which was then blocked with Odyssey Blocking Buffer (LiCor), or 5% fat-free milk. Following antibodies were used: anti-K48-linkage Specific Polyubiquitin (D9D5, Cell Signaling, #8081), anti-actin (Abcam ACTN05(C4), or Cell Signaling 8H10D10 Cat #3700), anti-Bcl-X_L (Cell Signaling #2764), anti-Bcl-2 (Dako, # M0877), and anti-Mcl-1 (BD Pharmigen, # 559027), anti-Puromycin (clone 12D10, EMD Millipore cat #MABE343), HRP-conjugated anti-mouse IgG (Cell Signaling #7074), HRP-conjugated anti-rabbit IgG (Cell Signaling #7076), IRDye800CW labelled anti-mouse-IgG (Licor, #926-68070), Alexa680-labeled anti-rabbit-IgG (Invitrogen, #A21076). Bands were revealed using SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) followed by imaging using a CCD camera (GelDoc (Bio-Rad) or Azure c600 instruments) or on Odyssey scanner (LiCOR) using auto-exposure settings.