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Fig. 5. The CaSR associates with RAMP1 and RAMP3. COS7 cells were transfected with SEP-CaSR in combination with Myc-RAMP1 and HA-RAMP3 as indicated. 48 hours post-transfection, cells were lysed and 600 µg of proteins were immunoprecipitated with monoclonal anti-HA, anti-Myc or anti-GFP antibodies. (A) RAMP1 is present in CaSR immunoprecipitates. Western blot (W.B.) with anti-Myc antibody (to detect Myc-RAMP1) of total proteins (input, lanes 1 and 2) and of immunoprecipitates isolated by incubation with indicated antibodies (lanes 3-6). Myc-RAMP1 was retrieved in anti-GFP immunoprecipitates prepared from COS7 cells expressing both the SEP-CaSR and Myc-RAMP1 (lane 3) but not when the anti-GFP antibody or the SEP-CaSR coding plasmid were omitted (lanes 4 and 5 respectively), indicating that the binding of Myc-RAMP1 occurs in a CaSR-dependent manner. Immunoprecipitation using anti-Myc antibodies indicates the expected size of immunoprecipitated Myc-RAMP1 (lane 6). (B) RAMP3 is present in CaSR immunoprecipitates. Immunoblot using anti-HA antibody (to detect HA-RAMP3) of total proteins (input, lanes 1 and 2) and of immunoprecipitates (lanes 3-5). RAMP3 was retrieved in anti-GFP immunoprecipitates prepared from COS7 cells expressing both the SEP-CaSR and HA-RAMP3 (lane 3) but not when the SEP-CaSR plasmid was omitted (lane 4), indicating that the binding of the HA-RAMP3 occurs in a CaSR-dependent manner. A positive control immunoprecipitate using anti-HA antibodies was loaded to indicate the expected size of HA-RAMP3 (lane 5). (C) The CaSR is present in RAMP3 precipitates. Western blot using anti-GFP antibody (to detect SEP-CaSR) of total proteins (input, lanes 1 and 2) and immunoprecipitates (lanes 3-5). The CaSR was retrieved in anti-HA immunoprecipitates prepared from COS7 cells expressing both the SEP-CaSR and HA-RAMP3 (lane 3) but not when HA-RAMP3 plasmid was omitted (lane 4), indicating that the binding of the CaSR occurs in a RAMP3-dependent manner. Immunoprecipitation using anti-GFP antibodies indicates the expected size of immunoprecipitated SEP-CaSR (lane 5). The monomeric and dimeric SEP-CaSR forms are indicated.





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