A. Schematic diagram showing the location of GPR56 shRNA constructs (2, 3, black arrows) against GPR56 transmembrane domains (rectangles) 2 and 4. The diamond indicates the G-proteolytic site. B. GPR56 mRNA expression by RT-qPCR in silenced C2C12s. Both shRNA2 and 3 effectively silenced the expression of GPR56. C. Western blot of GPR56, MyoD, and myogenin proteins in silenced C2C12 cells. D. Myosin Heavy Chain staining in GPR56-silenced cultures shows decreased myotube formation in GPR56 shRNA2 and 3 silenced cells. Scale bar = 50 µm. E. Fusion is decreased in GPR56-silenced cells at day 5 following differentiation. Un, uninfected; scr, scrambled oligo. * p < 0.01. F. Myotube size is decreased in GPR56-silenced cells at day 5 following differentiation. *p < 0.01. un, uninfected; scr, scrambled oligo. G. Schematic showing full-length and truncated GPR56. Diamond = G proteolytic site. Rectangles = transmembrane domains. H. Luciferase reporter assays in HEK293 cells of full-length (mGPR56, black diamond) or truncated (tGPR56, gray squares) GPR56 with luciferase reporter constructs driven by serum response element (SRE) or NFAT response element (NFAT-RE). GPR56 induces signaling from both SRE and NFAT-RE. * p<0.05. # p<0.001. n=3.