Fig. 4. Role of ARNO in CaSR-induced PM ruffling. (A) ARNO(E156K) impairs CaSR-induced PM ruffling. CaSR-HEK cells expressing GFP, Myc-ARNO(E156K) or Myc-WT-ARNO (cells shown in C) were stimulated with 5 mM CaCl_2, for 10 minutes, fixed and stained for Myc-ARNO (using 9E10 anti-Myc monoclonal antibody followed by a Cy2-labelled anti-mouse Ab) (green), and F-actin (red). Arrows indicate the cells that did not change shape. Arrows point to transfected cells whose morphology was unaffected and arrowheads point to ruffles. The micrographs are representative of three independent experiments. Bar, 10 µm. (B) Quantification of CaSR-induced PM ruffling illustrated in A. ^*P0.001. NS, no significant difference between tested conditions (P>0.05); n=3. (C) Myc-ARNO-expressing CaSR-HEK cells were challenged or not with 5 mM CaCl₂ for 10 minutes, fixed and stained for Myc-ARNO and F-actin as described in A. Bar, 5 µm. Note ARNO relocation to protrusions following CaSR stimulation.