Western Blot Analysis of Complement Proteins

Western blotting was performed as previously described with modifications^{24 (link)}. Cell proteins were isolated using 1 × RIPA buffer with protease inhibitor and phosphatase inhibitor. The proteins were resolved by electrophoresis in a 12% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (GE Healthcare). The membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20. Mouse monoclonal anti-C3b (Thermo scientific), mouse monoclonal anti-properdin (Abcam), rabbit polyclonal anti-CD55 (Santa Cruz biotechnolofy), rabbit polyclonal anti-CD59 (Abcam), rabbit polyclonal anti-CD46 (Santa Cruz Biotechnology), mouse monoclonal anti-ERK(1/2) (Bioss Antibodies Inc., Woburn, MA), rabbit polyclonal anti-phospho ERK (Bioss Antibodies Inc.), rabbit polyclonal anti-AKT (Bioss Antibodies Inc.), rabbit polyclonal anti-phospho AKT (Cell Signaling Technology, Beverly, CA) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies. HRP-conjugated anti-rabbit or anti-mouse antibodies (Bethyl Laboratories Inc., Montgomery, TX) were used as secondary antibodies. The results were visualized using an ECL detection reagent (Bio-Rad, Hercules, CA).