FIGURE 3.

gC1qR was identified as a binding protein for TLQP-21. A, biotinylated TLQP-21 was conjugated to Sulfo-EMCS and applied to membrane fractions of rat brain. Samples were resolved using Tricine-PAGE and visualized by streptavidin-horseradish peroxidase. A clear band at ∼30 kDa (indicated by an arrow) was observed following cross-linking using biotinylated TLQP-21, but not in the membrane control lane using unconjugated cross-linker. B, membrane fractions of rat forebrain were applied to monomeric avidin column attached to TLQP-21. Proteins were eluted and resolved on SDS-PAGE followed by silver staining. A ∼30-kDa band (arrow) was apparent in the elutant from the TLQP-21-attached column, but not in monomeric avidin column only (Negative control). C, the gC1qR protein was eluted with TLQP-21 following monomeric avidin-based affinity chromatography. D, the gC1qR protein was expressed by both brain- and spinal cord-derived microglia and bone marrow-derived macrophages. E, the ∼30-kDa band was excised, proteins were in-gel digested with trypsin, and the resulting peptide mixture was analyzed by nano-LC-MS/MS. MS/MS spectra show three unique peptides (E1–E3) which have sequence identities to gC1qR.