Pierce power blotter

Cell lysates were prepared by incubating Nonidet P-40 cell lysis buffer (Amresco, Solon, OH, USA) with cells for 30 min before high-speed centrifugation. Lysates were collected and ran in 15% gels using vertical gel electrophoresis. Protein content was transferred from gels to nitrocellulose blots using the Pierce Power Blotter (Thermo Fisher Scientific). GAPDH served as a loading control. After primary antibody staining, secondary antibodies conjugated to fluorochromes were used for visualizing protein bands on the Odyssey imaging system (LI-COR, Lincoln, NE, USA). Image Studio 5.2 software (LI-COR) was used to calculate relative fluorescence intensities.

Cell lysates were prepared or immunoprecipitation (IP) samples were eluted using Loading buffer, subsequently followed by heating at 95 °C for 5 min. SDS-PAGE was then implemented to separate proteins by molecular weight using the Mini-PROTEAN 3 electrophoresis module apparatus (Bio-rad). The gels were then subsequently transferred using the Pierce Power Blotter (Thermo Scientific), with reagents and instructions used from the Trans-Blot® Turbo™ RTA Mini Nitrocellulose Transfer Kit (Bio-rad), for 11 min, at a constant of 25 V. Membranes were then incubated with the appropriate Primary antibody overnight at 4 °C and then a minimum of 3 h at 4 °C with the appropriate Secondary antibody. Protein expression was analysed and detected using the Western Lightning® Plus-ECL, Enhanced Chemiluminescence Substrate (PerkinElmer) and G:BOX Chemi XX6 gel doc system (Syngene). A list of antibodies is provided as a Supplementary Table.

Protein extracts were prepared using a RIPA buffer (50 mM Tris/HCl (pH 8.0), 0.1% SDS, 0.5% sodium desoxycholate (all from Sigma-Aldrich), 150 mM NaCl (Carl Roth, Karlsruhe, Germany), and 1% Triton X-100 (Roche, Penzberg, Germany)) with 5% protease inhibitor (Sigma-Aldrich). Protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were diluted to equal concentrations with 1× Roti^®-Load 1 (Carl Roth). SDS-PAGE was performed following standard protocols. The Pierce™ Power Blotter (Thermo Fisher Scientific) was used for semi-dry protein transfer to PVDF membranes (Pall, Port Washington, NY, USA). Membranes were blocked with 5% non-fat dried milk (AppliChem, Darmstadt, Germany)/TBS-T (40 mM Tris/HCl (pH 7.6), 273 mM NaCl, 0.1% Tween^® 20 (Sigma-Aldrich)) for 1 h at room temperature. Incubation with primary antibodies was performed in blocking solution overnight at 4 °C, and incubation with secondary antibodies was performed in TBS-T for 1 h at room temperature. Immunoblots were developed with SuperSignal West Pico or Femto Chemiluminescent Substrates (Thermo Fisher Scientific), and signals were detected on a ChemiDoc Touch Imaging System (Bio-Rad). Bands were quantified using Image Lab v6 (Bio-Rad). The antibodies and the concentrations used are listed in Supplementary Table S2.

For the western blotting of NF-κB p65 and phospho-p65, 20 μg of whole cell lysates was used for SDS polyacrylamide gel-running, followed by transfer to a polyvinylidene difluoride (PVDF) membrane using Pierce Power Blotter (Thermo Fisher Scientific) at 110 V (80 mA) for 80 min. The membrane was incubated in 5% skim milk/0.1% TBS-Tween 20 at room temperature for 1 h, followed by incubation with a 1:1,000 final diluted NF-κB p65 (#8242, Cell Signaling Technology) and phospho-p65 antibody (Ser536, #3033, Cell Signaling Technology) and then an anti-rabbit secondary antibody conjugated with horseradish peroxidase (#7074, Cell Signaling Technology). The control for loading was assessed using a β-actin antibody (at a dilution of 1:1,000, SC-47778, Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States).