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Fig. 2. Cell-type-specific CaSR surface expression. (A) SEP-CaSR expression at the plasma membrane. Surface CaSR was labelled without permeabilisation in HEK293 cells expressing SEP-CaSR using a rabbit anti-GFP antibody followed by a Cy3 anti-rabbit antibody. Colocalisation of the SEP-CaSR fluorescence (green) and surface-labelled SEP-CaSR (Cy3, red) is revealed in yellow. A corresponding transmitted light image is shown to highlight the localisation of SEP-CaSR at the plasma membrane (arrowheads). Representative micrographs of five independent experiments are shown. (B) Confocal images showing differential CaSR distribution in HEK293 and COS7 cells expressing SEP-CaSR. Surface CaSR was labelled without permeabilisation using a rabbit anti-GFP antibody followed by a Cy3 anti-rabbit antibody. The overlay of total (green, left) and surface SEP-CaSR (Cy3, red, centre) is revealed in yellow (right). Arrowheads indicate the plasma membrane. A corresponding transmitted light image is shown for COS7 cells to highlight the lack of staining at the plasma membrane. Representative micrographs of three independent experiments are shown. (C) Differential CaSR cell surface expression assessed by surface biotinylation experiments. Surface proteins from COS7 and HEK293 cells transfected with SEP-CaSR were isolated by surface biotinylation as described in the Methods and the total and surface CaSR populations revealed by western blotting using an antibody against GFP. Representative blots of four independent experiments are shown. (D) Cell-type-restricted RAMP mRNA expression. RT-PCR amplifications of RAMP1, RAMP2 and RAMP3 mRNA were performed on samples from HEK293 and COS7 cell cultures and resolved on an agarose gel. A similar expression pattern was obtained in all the preparations tested (three independent experiments). Before PCR amplification, samples were (+) or not (–) submitted to reverse transcriptase (RT) treatment to estimate DNA contamination. GAPDH was used as an internal control and sizes of expected products are indicated. Bar, 5 µm (A); 10 µm (B).
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