Westernbright quantum kit

Total protein extraction, purification of histones and Western blot analysis were performed as previously described [33 (link)]. Briefly, cells were incubated for 30 minutes on ice in RIPA-buffer (150 mM NaCl, 1% Triton X-100, 0.5% desoxycholate, 1% Nonidet P-40, 0.1% SDS (sodium dodecyl sulfate), 1 mM EDTA, 50 mM TRIS (pH 7.6)) containing 10 µL/mL protease inhibitor cocktail (#P-8340, Sigma Aldrich). Histones were extracted by a modified published protocol employing sulphuric acid extraction and TCA-precipitation [34 (link)]. Concentrations of total protein and histones were determined by BCA protein assay (Thermo Fisher Scientific, Carlsbad, CA, USA). Subsequently, total cell proteins (50 µg) or extracted histones (2 µg) were separated by SDS-PAGE (total proteins 10% gels, histones 15% gels), transferred to PVDF membranes (Merck Millipore, Berlin, Germany) and were incubated with primary antibodies at RT for 1 h or 4°C overnight (see Table S2) following blocking with 5% non-fat milk in TBST (150 mM NaCl, 10 mM TRIS, pH 7.4 and 0.1% Tween-20). For signal detection membranes were incubated with a suitable horseradish peroxidase-conjugated secondary antibody (see Table S1) at RT for 1 h and signals were visualized by Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Munich, Germany) and WesternBright Quantum kit (Biozym, Hessisch Oldendorf, Germany).

Total protein was extracted by lysis for 30 min on ice in RIPA buffer pH 7.6 (150 mM NaCL, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 50 mM Tris pH 7.6, 1% protease inhibitor cocktail (#P8340) and 1% phosphatase inhibitor (#P0044, both Sigma-Aldrich). Determination of protein concentrations and western blot analysis of whole-cell extracts was performed as described previously [88 (link)]. Primary antibodies were used against NRF2 (1:1000, ab62352; Abcam, Cambridge, UK), KEAP1 (sc-365626), p62/SQSTM1 (sc-28359), YAP (sc-398182; all 1:1000; Santa Cruz Biotechnology, Heidelberg, Germany), NF-κB p65 (#610868) and IKKα (#556532; both BD Bioscience, San Jose, CA, USA, 1:1000). Secondary antibodies were HRP-conjugated goat-anti-mouse or goat-anti-rabbit antibodies (1:5000, sc-2005 or sc-2004, Santa Cruz Biotechnology, Heidelberg, Germany). Anti-α-Tubulin (1:10000, B-512, Sigma-Aldrich) was used as loading control. Expression levels were visualised by WesternBright Quantum Kit (Biozym, Hessisch Oldendorf, Germany) or Super Signal West Femto (Thermo Fisher Scientific, Waltham, MA, USA) on the Li-COR C-DiGit Blot Scanner using Image Studio Digits 4.0 software (LI-COR Biosciences, Lincoln, NE, USA).

UCC were treated for 72 h with cell line-specific half maximal inhibitory concentration (IC₅₀) doses of PLX before cell lysates were prepared. Immunoblot analysis was performed with whole cell extracts as described in [8 (link)]. Antibodies for target detection and secondary antibodies are listed in Table S1. Targets were visualized by SuperSignal™ West Femto (Thermo Scientific, Rockford, IL, USA) and WesternBright Quantum kit (Biozym, Hessisch Oldendorf, Germany).
Immunocytochemistry was performed as described in detail elsewhere [32 (link)]. Briefly, cells were treated on coverslips with the indicated doses, fixed with formaldehyde, permeabilized, and incubated in blocking solution. Cells were stained by the application of 50 μL of primary antibody solution per coverslip (Table S1) and incubated at 4 °C overnight. After washing, secondary antibodies were likewise added at room temperature for 1 h. After washing, the nuclei were counterstained with DAPI (4′,6-Diamidin-2-phenylindol). Finally, mounting medium (Dako, Santa Clara, CA, USA) was added. Images were taken by means of an Olympus Fluoview FV-1000 microscope (Hamburg, Germany), kindly provided by the Center for Advanced Imaging, Heinrich Heine University (Cai, numerical aperture 1/20, 60× objective). Images were processed using the Fluoview FV-1000 software, version 3.1.