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Fig. 1. Functional characterisation of SEP-CaSR. (A) Schematic representation of super-ecliptic pHluorin-tagged calcium-sensing receptor (SEP-CaSR). The plasma membrane and the flytrap domain are indicated. (B) Live confocal imaging of typical SEP-CaSR fluorescence distribution in two cultured HEK cells transfected with SEP-CaSR. Note that fluorescence is mainly visible at the plasma membrane. SEP-CaSR surface expression was assessed by monitoring fluorescence intensity in response to pH changes: reducing extracellular pH from pH 7.4 (Control, Ctl) to pH 6 causes a decrease in fluorescence as surface SEP-CaSR is eclipsed. Surface fluorescence is restored following a rinse with the pH 7.4 control solution. Application of a solution at pH 7.4 containing NH4Cl (50 mM), which rapidly equilibrates cellular pH levels, causes a sharp increase as all the SEP-CaSR fluorescence in the cell is revealed. The intracellular fluorescence disappears following a rinse with the control solution. (C) Immunoblot of SEP-CaSR. Cell extracts (
20 µg) from HEK cells expressing pRK5 (empty vector, as a control), pRK5-CaSR or pRK5-SEP-CaSR were run on an 8% polyacrylamide gel and probed with anti-CaSR ADD antibody (left panel) or with anti-GFP antibody (right panel). Position of molecular mass markers is indicated and arrowheads indicate the putative monomeric and homodimeric forms of SEP-CaSR. (D) Intracellular calcium concentration is increased by SEP-CaSR activation. HEK293 cells were transfected with SEP-CaSR (green) and loaded with the calcium-sensitive dye Fura Red (red). Merged areas appear yellow. The corresponding transmitted light image is shown. (E) Graph of the time courses of fluorescence from the regions selected in the fluorescence and transmitted light images in D. Stimulation with 5 mM CaCl2 causes oscillations in the Fura Red fluorescence intensity, which corresponds to [Ca2+]i oscillations, in the SEP-CaSR-expressing cell. The data are representative of three independent experiments. Bar, 10 µm.
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