Fig. 2. -arrestin 1 is required for CaSR-induced PM ruffling. (A) Expression of a dominant negative form of -arrestin 1 impairs CaSR-induced PM ruffling. Representative pictures of HEK cells expressing SEP-CaSR in combination with dominant negative -arrestin 1 (319-418) or wild-type (WT) -arrestin 1. HEK cells were co-tranfected with SEP-CaSR (1 µg) and excess (4 µg) dominant negative -arrestin 1 (319-418), wild-type (WT) -arrestin 1 or CAT (as a control plasmid). Cells were stimulated with CaSR agonists (5 mM CaCl_2, 10 µM NPS R-467) for 10 minutes, fixed and stained for SEP-CaSR (green), and F-actin (red). Arrowheads indicate ruffles. The micrographs are representative of three independent experiments. (B) Histogram (expressed as mean±s.e.m.) of CaSR agonist-induced PM ruffling in the conditions presented in A. ^*P0.001 compared with control plasmid-expressing cells stimulated with agonists (n=3). (C) siRNA-mediated silencing of -arrestin 1 impairs CaSR-induced PM ruffling. HEK cells were transfected with a siRNA duplex targeting -arrestin 1 (or a siRNA control) together with SEP-CaSR. 50 hours post-transfection, cells were stimulated with agonists, imaged and scored. Arrows indicate transfected cells whose morphology was unaffected and arrowheads point to the ruffles. (D) Quantification of data represented in C. ^*P0.001 compared with control siRNA-expressing cells stimulated with agonists. (n=3). (E) Protein samples prepared in parallel to the ruffling experiments from cells transfected simultaneously with CaSR and siRNA, described in C and D, were probed with CaSR (top panel) and pan -arrestin (A1CT, bottom panel) antibodies, respectively. (F) Pan -arrestin (A1CT) blot of cells transfected with control or -arrestin 1 siRNA alone showing 60% knockdown of -arrestin 1 (see text for details).