P0049

Maxisorp microtiter plates (96-well, Invitrogen) were coated with rabbit anti-human C3c (Dako #A0062) overnight, at 4 °C. After coating, plates were blocked with Quench for 1 h at 37 °C, then incubated with 50 µl/well with supernatant or lysate of WT or mutant C3 transfected HEK293 cells, diluted in Quench supplemented with 10 mM EDTA, for 1 h at 37 °C. C3 was detected using goat anti-human C3 antibody (Quidel #A304) and HRP-conjugated rabbit anti-goat immunoglobulins (Igs) (Dako #P0049) for 1 h at RT.

Cells were lyzed with RIPA buffer (Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Sigma‐Aldrich) and sonicated. The DC protein assay (Bio‐Rad, Hercules, CA) was used to quantify protein concentrations and equal amounts of protein (20 μg/lane) were subjected to SDS gel electrophoresis on stain‐free TGX mini‐PROTEAN precast gels. Before protein transfer, hydrogels were put under UV to activate stain‐free trihalo components in the gel, and gel images were taken for total protein quantification and normalization as described previously (Ladner et al. 2004). After activation, protein transfer to a nitrocellulose membrane was performed using the semidry Transblot Turbo system (Bio‐Rad). Membranes were blocked in 5% skimmed milk in Tris‐buffered saline + 0.1% Tween 20 and incubated overnight with primary antibodies: mouse monoclonal to αSMA (Clone 1A4, 0.28 μg/mL; Dako) and goat polyclonal to collagen type I (sc‐8783, 2 μg/mL; Santa Cruz Biotechnology). Next day, after three washes with TBST, membranes were incubated with goat‐anti‐mouse HRP (P0447, 1 μg/mL; Dako) or rabbit‐anti‐goat HRP (P0049, 0.5 μg/mL; Dako) for 1 h at RT. Protein bands were visualized with chemiluminescence (ECL, Thermo Fisher Scientific) and a ChemiDoc imaging system (Bio‐Rad). Image analysis was performed with ImageJ version 5.1 (Schindelin et al. 2012).