Albumin bovine 5

Harvested cells were washed with ice-cold PBS and lysed using RIPA lysis buffer (89900, Thermo Fisher Scientific, USA) supplemented with 1 mM PMSF (36978, Thermo Fisher Scientific, USA). The protein concentration was measured using the Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher Scientific, USA), and equal amount (40 µg) of the lysates were separated by 10% SDS-PAGE. The proteins were electro-transferred to PVDF membranes, and blocked with 5% albumin bovine V (10735086001, Roche, USA) in Tris-buffered saline containing 0.5% Tween 20 (TBST buffer) at room temperature for 2 h. The membranes were then incubated overnight with the specific primary antibodies against CD147 (#13287), PI3K (#4249), p-PI3K (#4228), PDK1 (#5662), p-PDK1 (#3438), AKT (#9272), p-AKT (#4058), GLUT-1 (#12939) and GAPDH (#8884; all from Cell Signal Technology, USA). The specific protein bands were visualized using the SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34577, Thermo Fisher Scientific, USA) and Tanon 5500 Chemiluminescence Detection System (Tanon, Shanghai, China).

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).