Coomassie blue dye

Fetal Bovine Serum (FBS) and Phosphate Buffered Saline (PBS) were purchased from Hyclone™, ThermoFisher Scientific, Waltham, MA, USA. HEPES, EDTA, EGTA, TEMED, isopropanol, aprotonin, pepstatin, phenylmethanesulfonyl fluoride (PMSF), leupeptin, sodium fluoride (NaF), sodium orthovanadate (Na₃VO₄), diethyldithiocarbamate (DDC), doxorubicin, etoposide, fludarabine, 5-flouroUracil (5-FU), cisplatin, dimethyl-9,9’-biacridinium dinitrate (Lucigenin), N-acetyl-cysteine (NAC) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Venetoclax (VEN) was purchased from Medchem Express LLC. Streptomycin–penicillin and l-glutamine were purchased from Gibco. Coomassie Blue dye and bovine serum albumins (BSA) were purchased from ThermoFisher Scientific. Methanol and sodium dodecyl sulphate (SDS) were purchased from Merck, Kenilworth, NJ, USA. Sucrose and 30% acrylamide/bis solution were purchased from Bio-Rad. Goat anti-mouse and anti-rabbit IgG horseradish peroxidase conjugated secondary antibodies were purchased from Pierce, TX, USA. Protein A agarose beads were purchased from Santa Cruz, TX, USA. Chloromethyl-2-,7-dichlorofluorescin diacetate (DCFDA) and MitoSox™ were purchased from Molecular Probes, Thermo Fisher Scientific.

C5 (human or mouse; 2 μg) was resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing and non‐reducing conditions on 7·5% PAGE gels, alongside commercially sourced human C5 (Complement Technology Inc, Tyler, TX). Gels were stained with Coomassie Blue dye (ThermoFisher). For Western blotting, 1 μg of C5 was resolved by SDS–PAGE under reducing and non‐reducing conditions as above, then electrophoretically transferred onto 0·45‐μm nitrocellulose membrane (GE Healthcare). After transfer, non‐specific sites on the membrane were blocked with 3% bovine serum albumin (BSA) in PBS containing 0·1% Tween‐20 (PBS‐T). After washing in PBS‐T, the membrane was incubated with the primary antibody; RO7112689 (at 1 μg/ml in 3% BSA PBS‐T) or polyclonal (goat) anti‐human C5 (CompTech, Texas, USA; A220; 2 μg/ml in 3% BSA PBS‐T). After washing, bound antibodies were detected by incubation with donkey anti‐human IgG‐horseradish peroxidase or rabbit anti‐goat IgG‐horseradish peroxidase conjugate (Jackson ImmunoResearch, W Baltimore Pike, West Grove, USA; 709‐036‐149, 305‐035‐045) at 1 : 5000 in PBS‐T. After washing, the blot was developed with enhanced chemiluminescence (GE Healthcare) and visualized by autoradiography.