Mouse anti c5ar

50 μg of whole cell lysate in 1x PBS were separated on a 4–20% gradient Mini-PROTEAN TGX precast gradient gel (Bio-Rad) under reducing conditions and transferred to a 0.2 μm Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA, United States). After washing, the membrane was blocked for 1 h with 10% normal donkey serum (NDS) in PBS, followed by probing with goat anti-rat GAPDH (R&D Systems, Minneapolis, MN, United States), followed by a 1:5000 donkey anti-goat IRDye 680 secondary antibody. The membrane was blocked with 10% normal goat serum (NGS) in PBS, followed by probing with rabbit anti-C3aR (Bioss Inc., Woburn, MA, United States) or Mouse anti-C5aR (Hycult Biotech., Plymouth Meeting, PA, United States), followed by a 1:5000 goat anti-rabbit IRDye 800 secondary antibody. The membrane was imaged using a LICOR Clx Odyssey imaging system using both 680 and 800 nm fluorescent channels.

Primary antibodies used for assays: Goat anti-rat C3 IgG (MP Biomedicals, Santa Ana, CA, United States), Chicken anti-C3/C3a (Abcam, Cambridge, MA, United States), Rabbit anti-rat C9 (generously provided by Professor Paul Morgan, Cardiff, United Kingdom), goat anti-human C1q (Complement Technology, Inc., Tyler, TX, United States), mouse anti-NeuN (EMD Millipore, Temecula, CA, United States), chicken anti-NeuN (EMD Millipore), mouse anti-GFAP (Sigma-Aldrich, St. Louis, MO, United States), chicken anti-GFAP (Abcam), goat anti-Iba1 (Abcam), rabbit anti-C3aR (Bioss Inc., Woburn, MA, United States), Mouse anti-C5aR (Hycult Biotech., Plymouth Meeting, PA, United States). Secondary antibodies included goat anti-mouse horseradish peroxidase (HRP) (Sigma-Aldrich), goat anti-chicken IgY HRP (Genway Biotech, San Diego, CA, United States), goat anti-rabbit HRP (Sigma-Aldrich), rabbit anti-goat HRP (Thermo Fisher Scientific, Grand Island, NY, United States), biotinylated goat anti-mouse IgG (H + L), biotinylated goat anti-chicken IgG (H + L) (Vector Laboratories, Burlingame, CA, United States), biotinylated goat anti-rabbit IgG (H + L) (Bioss Inc), donkey anti-goat IgG (H + L) Alexa Fluor (AF) 488/568, goat anti-chicken IgG (H + L) AF 488, goat anti-mouse IgG (H + L) AF 568, and goat anti-rabbit IgG (H + L) AF 405/488 (Life Technologies, Grand Island, NY, United States).