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. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: Peptides. 2012 Mar 30;35(2):202–211. doi: 10.1016/j.peptides.2012.03.020

Figure 3. Detection of human CLR and RAMP1 before and after stimulation with α-CGRP.

Figure 3

HEK cells were transiently transfected with empty vector (control) or human FLAG-CLR and human myc-RAMP1. The specificity of anti-rat CLR (RK11) and RAMP1 (9891) antibodies was determined by immunofluorescence and confocal microscopy. 3A, 3C: In unstimulated HEK-human CLR•RAMP1 cells, CLR (RK11, FLAG (M2)) and RAMP1 (9891, c-Myc) were detected at the cell-surface (upper panel, arrowheads). Incubation with human α-CGRP (100 nM, 30 min) caused internalization of both CLR (RK11, FLAG (M2)) and RAMP1 (9891, c-Myc) to intracellular vesicles (lower panel, arrows). Thus, RK11 and 9891 both cross-react with activated CGRP receptor components. 3B, 23D: In HEK cells transfected with empty vector (control) no signal for CLR (RK11, FLAG (M2)) or RAMP1 (9891, c-Myc) was detected indicating specificity of RK11 and 9891 antibodies. Pre-adsorption with respective peptides for RK11 and 9891 similarly abolished all staining. Scale bar = 10 μm.