Cbmab s1587 cq

Adult 2 month old SRPX2^+/Y and SRPX2^FLAG/Y mice were anesthetized and perfused with phosphate buffer saline (PBS). Brains were dissected and homogenized in lysis buffer containing 50 mM Tris, pH 7.5, 1% Triton X-100, 150 mM NaCl, 10% glycerol, and 2 mM PMSF using a Glas-Col homogenizer at 10 mL lysis buffer per gram wet weight. Brain homogenates were incubated at 4 °C for 30 min and then centrifuged at 1000 x g for 10 min, and 21,000 x g for 30 min at 4 °C. For co-immunoprecipitation, the supernatant was further incubated with anti-FLAG M2 magnetic beads (Sigma-Aldrich, Cat. no. M8823-1ML) for 3 hours at 4 °C. Magnetic beads were extensively washed with lysis buffer and TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.4). Protein complexes were eluted with 100 μL of 3X FLAG peptide (200 ng/μL) according to the manufacturer’s protocol (Sigma-Aldrich), and the eluates were blotted with anti-FLAG M2-peroxidase (HRP) (Sigma-Aldrich, Cat. no. A8592-0.2mg), anti-C1Q (Abcam, Cat no. ab182451), and anti-C3 (MP Biomedicals, Cat. no. 0855730) antibodies. For checking endogenous SRPX2 expression in the SRPX2^FLAG/Y knock-in mouse, the brain was dissected into whole brain (WB), cortex (CX), and mid-brain (MD) before homogenization, and the lysates were blotted with anti-SRPX2 (Creative Biolabs, Cat. no. CBMAB-S1587-CQ) and anti-GAPDH (Novus Biologicals, Cat. no. NB600-502).