Extended Data Fig. 8|. Centrifugal pull-down analysis of fluorescently labelled arrestin mutants.

a, Arrestin mutants were mixed with rod outer segment membranes containing rhodopsin (Rho), phosphorylated rhodopsin (RhoP), opsin (Ops) or phosphorylated opsin (OpsP). Rhodopsin samples were illuminated (>495 nm, 15 s) to obtain activated rhodopsin (Rho*) and phosphorylated activated rhodopsin (Rho*P), and then all samples were centrifuged at 20,800g for 10 min. The supernatant was removed, and the pellets were solubilized in loading buffer. Samples were subjected to SDS–PAGE, and gels were stained with Coomassie blue. MW, molecular weight marker kDa. Arrestin migrates slower than rhodopsin or opsin (arrestin (A) and receptor (R) bands are indicated by arrows). As controls, samples of arrestin in buffer alone (NS, nonspecific pull-down in isotonic or low salt buffer) or rhodopsin alone (bkd, background) were centrifuged alongside the other samples. The total amount of arrestin present in each assay (2.25 μg) is indicated in the lanes marked ‘Arr’. Arrestin ‘cysless’ corresponds to the background construct for all fluorescently labelled arrestin mutants (C63A, C128S, C143A, W194F) and is functionally equivalent to native wild-type bovine arrestin-1⁶¹. Representative gels, cropped to show desired lanes, are shown. Experimental conditions: 1 μM arrestin, 10 μM receptor, 50 μl sample volume; 50 mM HEPES, 130 mM NaCl pH 7 (isotonic buffer) for samples containing rhodopsin, 50 mM HEPES pH 8.5 (low-salt buffer) for samples containing opsin, 20 °C. b, Arrestin bands were quantified by densitometry using the program GelQuant.NET v.1.8.2. Each band is expressed as the fraction of total arrestin that was present in each experiment, and bars represent averages from n = 2 (arr cysless), n = 5 (Rho* and Rho*P, L173F), and n = 4 (all other conditions) independent experiments ± s.e.m. Essentially no background density from ROS was present at the molecular-weight range of arrestin. All mutants showed some amount of nonspecific pull-down in the different buffer conditions. Note that this nonspecific pull-down is subtracted from the pull-down data reported in the main text. The fluorescent NBD-labelled arrestin mutants bound to the different receptor variants at similar levels as the cysless arrestin control (Ops < Rho* < OpsP < Rho*P).