Ab181147

The primary antibodies used included those recognising complement C4B (C4B; ab66791), C3b (ab181147, ab200999), C1q subcomponent subunit C (C1QC; ab75756) (Abcam, Cambridge, UK), β-actin (ACTB; BS6007M, Bioworld Technology), and profilin-1 (PFN1; NBP2-02577, Novus Biologicals, Littleton, Colorado, USA). Horseradish peroxidase-conjugated anti-mouse (ab6728, Abcam) or anti-rabbit (A120-1019, Bethyl Laboratories, Montgomery, Texas, USA) antibodies were used as secondary antibodies at an appropriate dilution. Blots were visualised using the Super Signal West Pico Chemiluminescent Substrate detection system (Pierce, UK), according to the manufacturer’s instructions. An antibody against glyceraldehyde phosphate dehydrogenase (GAPDH) (ADI-CSA-335, Enzo Life Sciences, Inc, Farmingdale, New York, USA) was used as an internal control. All experiments were performed at least three times. Band intensities were quantified using Image J software (National Institutes of Health, Bethesda, Maryland, USA).

The expression of C3 in the serum and CFHR5 on the cyto-membrane of red blood cells of SLE patients and healthy individuals, and in the MRL/lpr mice model and control MRL/MPJ mice were detected by western blot using specific primary antibodies. Forty µg of proteins were loaded on each lane of 8% SDS-PAGE gel; after separation, the proteins were transferred onto polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membranes. PVDF membranes were then blocked with 5% Albumin Bovine V (Roche, USA) in Tris-buffered saline containing 0.5% Tween 20 and incubated at room temperature for 2 hours. Next, membranes were incubated overnight with the specific primary antibodies of each protein, such as anti-C3 (ab181147), CFHR5 (ab175254), and GAPDH (ab9485, Abcam Biotechnology, Inc., Cambridge, UK) at 4^oC. At the second day, membranes were taken out and washed with washing buffer 3 times, each time for 5 min. Then membranes were incubated with corresponding secondary antibodies for 1 h at room temperature. After incubation, membranes were washed with washing buffer 3 times, each time for 5 min. After washing, proteins were visualized by chemiluminescence using Super Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, Massachusetts, USA) by a Tanon 5500 Chemiluminescence Detection System (Tanon, Shanghai, China).