Fig. 6.

Interaction of β-arr2 with AP-2 upon AngII stimulation releases FAK from β-arr2 and its constitutive inhibition over FAK. a HEK cells were transfected with HA-FAK and GFP or β-arr-GFP, serum starved and left unstimulated or treated with 100 nM AngII (10 min). IPs were performed using anti-GFP antibodies, and both IPs and lysates were immunoblotted for HA-FAK, pY397-FAK, and GFP. Data represent mean ± s.e.m. of the ratio of co-immunoprecipitated HA-FAK to immunoprecipitated GFP normalised to GFP in the lysate, and normalised to untreated cells set to 100% from three independent experiments (**P < 0.01, t test). b HEK-293 cells co-transfected with HA-AT_1AR, RlucII-FAK, and β-arr2-GFP₁₀ or β-arr2ΔAP-2-GFP₁₀ were untreated or stimulated with 1 µM AngII for 15 min and BRET measured. Graph represents mean ± s.e.m. of BRET normalized to BRET in untreated cells transfected with β-arr2-GFP₁₀ and set to 100% from three independent experiments (4–6 replicates for each condition per experiment). *P < 0.05; one-way ANOVA, Bonferroni. c Live cell imaging of GFP-FAK and mCherry-β-arr2ΔAP-2 upon AngII treatment. Confocal images of HEK-293 cells transfected with HA-AT_1AR, GFP-FAK (grey), and mCherry-β-arr2ΔAP-2 (grey) were acquired in absence of stimulation (basal) and 5 min after the addition of 100 nM AngII. Merged images are shown as GFP-FAK in green and mCherry-β-arr2ΔAP-2 in red. d HEK-AT_1AR cells were transfected with indicated plasmids, processed as in a and both IPs and lysates were immunoblotted for HA-FAK and GFP-β-arrs. Three independent experiments were quantified as in a. *P < 0.05, one way ANOVA. e HEK-AT_1AR cells transfected with β-arr2-GFP or β-arr2ΔAP-2-GFP were serum starved and unstimulated or stimulated with 100 nM AngII for the indicated time. Lysates were immunoblotted as indicated. Data represent mean ± s.e.m of pY397-FAK/FAK and phospho-Paxillin/Paxillin values (β-arr2-GFP: black circles, β-arr2ΔAP-2-GFP: red squares), normalized to untreated β-arr2-GFP-transfected cells and set to one from three independent experiments