Fig. 2.

FAK interacts with β-arrs. a IPs with HA antibodies were performed on lysates of HEK-293 cells transfected with HA-FAK and GFP or GFP-β-arr1 or GFP-β-arr2. Lysates and IP were immunoblotted for GFP-β-arrs and HA-FAK. b IPs with anti-myc antibodies were performed on lysates of HEK-293 transfected with HA-FAK, Myc-β-arr1, or Myc-β-arr2. Lysates and IP were immunoblotted for β-arrs and HA-FAK. c Yeast reporter strain HF7c was transformed with the indicated plasmids. Growth in the absence of histidine indicates interaction between β-arr2 and FAK fusion proteins. d Purified GST, GST-β-arr1, or GST-β-arr2 bound to glutathione-agarose beads (Coomassie staining) were incubated with HEK-293 lysates transfected with HA-FAK and immunoblotted with an anti-HA antibody. Input is 5% of cell lysate. e Purified GST, GST-β-arr1, or GST-β-arr2 bound to glutathione-agarose beads (Ponceau red staining) were incubated with HEK-293 lysates and immunoblotted for endogenous FAK. Input is 5% of cell lysate. f IP with rabbit anti-β-arr1/2 antibodies or rabbit IgG were performed on rat brain lysates. Lysates and IP were immunoblotted for FAK and β-arrs. BRET saturation experiments were performed on adherent HEK-293 cells (g) or HEK-293 in suspension (h) after co-transfection with a constant amount of plasmid for RlucII-FAK and increasing concentrations of β-arr1-GFP₁₀ or β-arr2-GFP₁₀ plasmids. mBRET values (β-arr1-GFP₁₀: red circles; β-arr2-GFP₁₀: black squares) are means ± s.e.m. of three independent experiments (three replicates for each condition per experiment). BRET₅₀ values were 6.89 ± 1.38 and 6.0 ± 1.09 for β-arr1-GFP₁₀ and β-arr2-GFP₁₀, respectively (g)