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Fig. 5. -arrestin 1 interacts and colocalises with ARNO. (A) Myc-WT-ARNO co-immunoprecipitates with Flag--arrestin 1. CaSR-HEK cells were transfected with plasmids encoding Myc-WT-ARNO ± Flag--arrestin 1. 48 hours post-transfection, cells lysates were prepared and subjected to immnoprecipitation using Flag beads (see Materials and Methods). Retained proteins were eluted with Laemmli's buffer and immunoblotted using anti--arrestins and ARNO antibodies. A total of the lysate (input) was run in parallel. (B) Catalytically inactive Myc-ARNO(E156K) co-immunoprecipitates with Flag--arrestin 1. (C) CaSR stimulation does not affect ARNO association with Flag--arrestin 1. Cells co-expressing WT-ARNO and Flag--arrestin 1 were stimulated with CaCl₂ for the indicated periods of time and Flag immunoprecipitation was performed as in A. Total ARNO and ARNO bound to Flag--arrestin 1 are shown. (D) Colocalisation of Myc-WT-ARNO and GFP–-arrestin 1 in protrusions. Transfected CaSR-HEK cells were challenged with 5 mM CaCl₂ for 1 or 10 minutes, fixed and stained for GFP--arrestin 1 (green), Myc-ARNO (using 9E10 Ab followed by a Cy5-coupled anti-mouse Ab, shown in blue) and F-actin (Alexa-Fluor-568–phalloidin, red). An inset showing a magnified view for the 1 minute stimulation highlights -arrestin 1 and ARNO colocalisation in protrusions. Bar, 5 µm. (E) GFP-ARNO, RFP--arrestin 1 and Flag-CaSR colocalise in protrusions. Transfected HEK cells were challenged with 5 mM CaCl₂ for 10 minutes, fixed and stained for GFP-ARNO (green), mRFP--arrestin 1 (red) and Flag-CaSR (detected by mouse anti-Flag Ab followed by a Cy5-coupled anti-mouse Ab, blue). A magnified view of the PM ruffling area is shown in the bottom panels. Merge areas appear in white. Bar, 5 µm.

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