Factor 1

To measure the cofactor activity of sCR1 in cleavage of C3b, ¹²⁵I-C3b (100,000 cpm/assay) in PBS was mixed with factor I (125 nM; MerckMillipore, Darmstadt, Germany), and sCR1 (180 nM) in the presence or absence of Ecb (0–2.0 μM). Mixtures (50 μl) were incubated for 15 min at 37°C, and after reducing the samples with β-mercaptoethanol (5 min, 94°C) aliquots were loaded onto a 10% SDS-PAGE gel. The gel was autoradiographed and cofactor activity was evaluated as the intensity of the C3b α’-chain analyzed with GelEval software (FrogDance Software, Dundee, UK).

The commercial CFH, factor I, and C3b were purchased from Merck (Kenilworth, NJ, USA). Factor H short consensus repeat (SCR) units (SCR1–4, SCR7, SCR11‐14, and SCR19–20) were produced by Gene‐Script Corporation (Piscataway, Nanjing, China). Antibodies used in this study included a goat anti‐human CFH polyclonal antibody (Merck), a mouse anti‐human CFH monoclonal antibody (US Biological, Salem, MA, USA), a rabbit anti‐human uromodulin polyclonal antibody (Biomedical Technologies Inc., Stoughton, MA, USA), a mouse anti‐human uromodulin monoclonal antibody (Cedarlane, Burlington, NC, USA), and species appropriate secondary antibodies (anti‐mouse IgG‐alkaline phosphatase conjugated antibodies and anti‐rabbit IgG‐alkaline phosphatase conjugated antibodies) from Sigma‐Aldrich (St. Louis, MO, USA). TRITC‐conjugated rabbit anti‐goat IgG (Zhongshan Biotech, Guangdong, China) and FITC‐conjugated rabbit anti‐mouse IgG (Abcam, Cambridge, UK) were also used when necessary.

The measurement of cofactor activity of cFH followed previous study.⁷ Briefly, the experiment was performed in fluid mixture 20 μL, which included cFH (0.5 μg, Merck, Kenilworth), factor I (50 ng, Merck), C3b (3 μg, Merck) and UMOD (8 μg). The mixture was incubated at 37℃ for 10, 20, 30 and 40 minutes, respectively. C3b and its cleavage products were detected by western blotting with a polyclonal anti‐C3c antibody (Dako Cytomation).