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Fig. 4. Internal and surface colocalisation experiments of CaSR with RAMPs. (A) COS7 were transfected with plasmids encoding SEP-CaSR and Myc-RAMP1, HA-RAMP2, HA-RAMP3 or CAT (as a control). 48 hours post-transfection, cells were stained in permeabilised conditions (see methods) for CaSR and RAMPs using respectively rabbit anti-GFP and mouse anti-HA or Myc antibodies followed by Cy5 anti-rabbit or Cy3 anti-mouse antibodies. Colocalisation regions of RAMPs (red) and CaSR (blue) appear in purple and are indicated by arrowheads. Images are representative of three independent experiments. (B) Surface colocalisation of CaSR and RAMP3. COS7 cells were transfected with either control plasmid (upper panels) or SEP-CaSR in the absence (middle panels) or presence (lower panels) of HA-RAMP3. Cells were incubated in non-permeabilised conditions with anti-GFP and anti-HA antibodies followed by Cy5 anti-rabbit and Cy3 anti-mouse antibodies to label surface CaSR and surface RAMP3 respectively (see Materials and Methods). Regions of colocalisation between surface CaSR (blue) and surface RAMP (red) appear purple and triple colocalisation areas (white) are indicated by arrowheads. Images are representative of three independent experiments. Bar, 10 µm.
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