Western Blot Analysis

Total protein was extracted using RIPA lysis buffer (R0020, Solarbio Technology, Beijing, China) containing Phosphatase Inhibitor and Protease Inhibitor Cocktail (Targetmol, MA, USA) at 4°C. About 10 µg of total protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane (Merck KgaA, Darmstadt, Germany). Following blocking with the PBS buffer containing 5% skimmed milk powder, the membranes were incubated with various primary antibodies respectively, overnight at 4°C. These antibodies include rabbit anti-human C5aR1 (Ls-B5559, dilution of 1:1000; Lifespan, Seattle, WA, USA), rabbit anti-human AKT (4691S, dilution of 1:1000; Cell Signaling Technology, MA, USA), rabbit anti-human p-AKT (4060S, dilution of 1:1000; Cell Signaling Technology, MA, USA), rabbit anti-human β-actin (YT0099, dilution of 1:5000; Immunoway, TX, USA), rabbit anti-human ERK1/2 (4695S, dilution of 1:1000; Cell Signaling Technology, MA, USA), rabbit anti-human p-ERK1/2 (4370S, dilution of 1:1000; Cell Signaling Technology, MA, USA), rabbit anti-human p38 (8690S, dilution of 1:1000; Cell Signaling Technology, MA, USA), rabbit anti-human p-p38 (4511S, dilution of 1:1000; Cell Signaling Technology, MA, USA), mouse anti-human STAT3 (9139T, dilution of 1:1000; Cell Signaling Technology, MA, USA), rabbit anti-human p-STAT3 (9145T, dilution of 1:1000; Cell Signaling Technology, MA, USA), rabbit anti-human p-SAPK/JNK (4668T, dilution of 1:1000; Cell Signaling Technology, MA, USA), mouse anti-human GAPDH (YM3029, dilution of 1:10000; Immunoway, TX, USA), rabbit anti-human CPD (NBP1-91447, dilution of 1:1000; Novus Biologicals, Centennial, CO, USA), rabbit anti-human CPM (NBP1-87403, dilution of 1:1000; Novus Biologicals, Centennial, CO, USA), rabbit anti-human CPN (Ls-c166993, dilution of 1:1000; Lifespan, Seattle, WA, USA) and rabbit anti-human CPZ (Ls-199274, dilution of 1:1000; Lifespan, Seattle, WA, USA). Then the membrane was washed with TBST three times and incubated with HRP-conjugated goat anti-rabbit (B0201, dilution of 1:10000; Immunoway, Texas, USA) or rabbit anti-mouse (B0101, dilution of 1:10000; Immunoway, Texas, USA) IgG antibody for 2 h at 37°C. The immunolabeled proteins were detected by chemiluminescence using the Chemiluminescent hRP substrate (WBULS0100, Merck KgaA, Darmstadt, Germany). Densitometric analysis was performed using ImageJ software, version 1.52a (National Institutes of Health, Bethesda, MD, USA).