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Fig. 3. Effect of CaSR stimulation on -arrestin 1. (A) Representative frames from live confocal microscopy experiments showing the subcellular redistribution of GFP--arrestin 1 in HEK cells co-expressing CaSR before (0 seconds) and after (30-300 seconds) activation by 5 mM CaCl₂. (B) HEK cells co-expressing GFP--arrestin 1 and CaSR (1 and 4 µg, respectively) were stimulated with 5 mM CaCl₂ for 2 or 10 minutes, fixed and stained for F-actin. Colocalisation of GFP--arrestin 1 (green) and F-actin (red) appears in yellow. Arrows indicate ruffles. Bar, 5 µm. Data are representative of four independent experiments. (C) The CaSR is constitutively associated with -arrestin 1. HEK293 cells were transfected with SEP-CaSR and Flag--arrestin 1 in the indicated combinations. 48 hours post-transfection, cells were stimulated with 5 mM CaCl₂ for different periods of time and lysed. 400 µg of resulting proteins were subjected to immunoprecipitation (IP) using sepharose beads coupled to Flag-M2 antibodies. CaSR present in the immunoprecipitate and total fractions of CaSR and -arrestin 1 (input) were assessed by immunoblotting (IB).

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