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. 2011 Jul 18;108(31):12925–12930. doi: 10.1073/pnas.1104821108

Fig. 1.

Fig. 1.

Identification of GPR56 ligand. (A–D) GPR56N-hFc binds specifically the putative ligand of GPR56 (green) in meninges and pial BM (A) and MFs (C). GPR56Ndel-hFc was used as a negative control (B and D). A and B are composite images combined into a single image. Nuclear counterstain was performed by Hoechst 33342 (blue). (Scale bars: A and B, 200 μm; C and D, 100 μm.) (E) SDS/PAGE analysis of purified GPR56 ligand(s) eluted from streptavidin affinity resin. The putative ligand of GPR56 is a large protein with a molecular weight of 160–260 kDa (arrowheads). (F) Summary of MS results from two independent purifications. Total peptides sequenced and proteins specifically identified by GPR56N are indicated. Collagen III appeared as the top candidate based on the high number of matched peptides. (G) The association of collagen III and GPR56N was confirmed by co-IP. Collagen III was specifically pulled down by GPR56N, but not GPR56Ndel. Conversely, only GPR56N was pulled down by anti-collagen III antibody, but not GPR56Ndel.