ERK2 binding to arrestin-1 and both non-visual arrestins is direct.

<p><b>A</b>. Active (phosphorylated at T183 and Y185 by MEK1) or inactive ERK2 (30 pmol) was pre-incubated with or without 30 pmol of indicated arrestin for 20 min at 30°C, then phosphorylated rhodopsin (50 pmol) was added and incubated in the light (to produce P-Rh*) in 0.1 ml for 5 min. Rhodopsin-containing membranes were pelleted through 0.2 M sucrose cushion and dissolved in SDS sample buffer. ERK2 in the pellet (1/300 of each sample) was quantified by Western blot using anti-ERK antibodies (Cell Signaling) and known amounts of purified ERK2 to generate calibration curve. Abbreviations: Arr1, visual arrestin-1, Arr2, arrestin-2, Arr3, arrestin-3. Representative blot is shown. <b>B</b>. Quantification of ERK2 binding to P-Rh*-associated arrestins. <b>C</b>. CNBr-activated Sepharose (30 µl) containing 9 µg of covalently attached active phosphorylated (without or with 1 mM ATP) or inactive ERK2 was incubated with 3 µg of indicated purified arrestin in 60 µl of binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM EGTA, 1 mM DTT) for 20 min at 30°C. The beads were washed twice with 1 ml of ice-cold binding buffer supplemented with 0.01 mg/ml BSA. Bound arrestins were eluted with SDS sample buffer and quantified by Western blot, where known amounts of respective arrestins were run alongside samples to generate calibration curves. Means ± SD of three independent experiments are shown in panels <b>B</b> and <b>C</b>.