Ponceau s

Cells were lysed by sonication using a Vibra Cell sonicator (Sonics & Materials, USA) and heated for 5 min at 95 °C. An equivalent of 3x10⁶ cells per lane was separated at 170 V on 10% polyacrylamide gels. After separation, samples were blotted onto nitrocellulose membranes (GE Healthcare Life Sciences, Germany). Uniform loading of gels was confirmed by Ponceau S (AppliChem, Germany) staining. Subsequently, membranes were blocked for 1 h with TBS-T (20 mM Tris–HCl [pH 7.6], 137 mM NaCl, 0.1% [v/v] Tween 20), containing 5% fat-free dry milk, and were washed afterwards thrice with TBS-T. Incubation with primary mouse monoclonal pZIP7 (S275/S276) [21] (link), ZIP7 (ProteinTech, United Kingdom) and β-actin (Cell Signaling Technology, Germany) antibodies was performed overnight at 4 °C at 1/1000 dilution in TBS-T, containing 5% BSA. Afterwards, membranes were washed thrice with TBS-T and incubated with either anti-rabbit-HRP (for β-actin and ZIP7) or anti-mouse-HRP (for pZIP7) (both from Cell Signaling Technology, Germany) secondary antibodies 1/2000 in TBS-T with 5% fat-free dry milk. After again washing thrice with TBS-T, immunodetection was performed using LumiGlo Reagent (Cell Signaling Technology, Germany) on LAS-3000 (Fujifilm Lifescience, Germany). Band density was determined with ImageJ software (NIH, USA).

The proteins were separated depending on their molecular weight in SDS-PAGE using 12.5% acrylamide gels. The proteins were transferred on a nitrocellulose membrane using semi-dry blot chamber. Subsequent to Ponceau S (AppliChem GmbH, Darmstadt, Germany) staining, unspecific binding was blocked by incubation in 5% skim milk powder diluted in PBS-T (137 mM NaCl, 2.7 mM KCl 8 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 0.1% Tween^®20, pH 7.4) for 1 h at RT. The membrane was quickly washed with PBS-T and incubated with actin monoclonal antibody (1:10,000, ACTN05(C4); Thermo Fisher Scientific, Waltham, MA, USA) or streptavidin-peroxidase conjugate (1:5000; Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS-T for 1 h at RT. Afterwards, unbound proteins were removed by washing three times with PBS-T for 5 min at RT on an orbital shaker. For the detection of actin, the membrane was further incubated with the secondary goat anti-mouse antibody-HRP (1:2500; Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at RT and washed three times with PBS-T. The peroxidase marked proteins were detected using Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and X-ray films (AGFA Health Care, Mortsel, Belgium).

TUBE pull-down assay was performed as previously described.^{35 (link)} Cells were lysed in buffer (50 mM NaCl, 20 mM Tris pH 7,5, 1% NP40, 5 mM EDTA, 10% glycerol) supplemented with a protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany) for 20 min on ice. In all, 1000 μg of protein were incubated overnight at 4 °C with 50 μl GSH-agarose beads (Sigma-Aldrich) linked to GST-TUBE. Afterward beads were washed with buffer. The precipitate was analyzed for ubiquitin expression by Western blotting using mouse IgG1 anti-ubiquitin (P4D1) (Santa Cruz Biotechnology) antibody and for interaction with NOXA and MCL-1 by Western blotting using rabbit anti-MCL-1 antibody (ENZO), and mouse anti-NOXA antibody (ENZO). To verify the protein amount per lane, the membrane was stained with Ponceau S (AppliChem GmbH, Darmstadt, Germany).