Fig. 3. RAMP1 and RAMP3, but not RAMP2, promote cell surface delivery of the CaSR. (A) Effect of RAMPs on CaSR surface expression assessed by surface biotinylation. COS7 cells were transfected with SEP-CaSR in combination with a control vector (encoding CAT) or vectors encoding RAMP1, RAMP2 or RAMP3. 48 hours post-transfection, the total and cell surface CaSR populations were determined as described in Fig. 2 legend and Materials and Methods. A representative western blot of four independent experiments is shown. (B) Densitometric analysis of the effect of RAMPs on CaSR surface expression measured by biotinylation assays as illustrated in A. Results are the mean±s.e.m. of four independent experiments. *P0.001 compared with levels in the control (Student's t-test). (C) Effect of RAMPs on CaSR surface expression assessed by immunofluorescence. COS7 were transfected with plasmids encoding SEP-CaSR and Myc-RAMP1, HA-RAMP2, HA-RAMP3 or CAT (as a control). Cells were then stained for surface CaSR using sequentially rabbit anti-GFP and Cy3 anti-rabbit antibodies. Cells were then permeabilised and Myc-RAMP1, HA-RAMP2 or HA-RAMP3 were labelled using mouse anti-Myc or anti-HA antibodies followed by Cy5 anti-mouse antibodies. Surface CaSR and RAMPs appear in red and blue respectively. A transmitted light image is shown for the control condition. Images are representative of three independent experiments. Bar, 10 µm.