Mouse monoclonal anti c3b

Western blotting was performed as described previously with modifications [32 (link)]. Cells or EVs were lysed in 1x RIPA buffer with protease inhibitors. The lysate was centrifuged and the supernatants collected. Mouse monoclonal anti-beta-actin (Sigma), mouse monoclonal anti-C3b (Thermo scientific), mouse monoclonal anti-properdin (Abcam), rabbit monoclonal anti-HSP70 (Abcam), rabbit polyclonal anti-CD59 (Abcam), rabbit polyclonal anti-CD55 (Santa Cruz Biotechnology), mouse monoclonal anti-CD63 (Santa Cruz Biotechnology), mouse monoclonal anti-CD81 (Santa Cruz Biotechnology), Rabbit polyclonal anti-CD46 (Santa Cruz Biotechnology), Rabbit polyclonal anti-Calnexin (Bioss Antibodies Inc.), and rabbit polyclonal anti-CD55 (Santa Cruz Biotechnology) were used.

Cells were seeded onto a microscopy cover glass in 24-well tissue culture plates at a density of 1 × 10⁵ cells/well. After culturing overnight, culture media was removed and washed with PBS. The cells were fixed with 4% paraformaldehyde and blocked with 3% bovine serum albumin (BSA) in PBS. Rabbit polyclonal anti-C5b-9 (Abcam, Cambridge, MA), mouse monoclonal anti-C3b (Thermo Scientific, Rockford, IL), or rabbit polyclonal anti-properdin (Bioss Antibodies Inc., Woburn, MA) were used as primary antibodies. Cells were incubated with a primary antibody, then incubated with Alexa Fluor-conjugated goat anti-rabbit or goat anti-mouse antibody (Invitrogen, Carlsbad, CA). Nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI). Cells were mounted with Vectashield^® (Vector Laboratories Inc., Burlingame, CA) and examined using an Eclipse E400 microscope (Nikon Instruments Inc., Melville, NY). Images were captured using a Nikon Digital site Fi3, and analyzed using NIS element BR.

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).