Phosphorylation of the rod cGMP-gated channel β-subunit by protein kinase(s) in the ROS soluble fraction. ROS membranes were hypotonically lysed, washed by centrifugation, and resuspended in either HEPES buffer or ROS lysate containing endogenous ROS soluble proteins. The phosphorylation reaction was initiated by addition of [γ-³²P]ATP, and the samples were incubated for 15 min at 30°C. The phosphorylated membranes were washed in HEPES and solubilized in CHAPS detergent. The cGMP-gated channel complex that bound to the PMc 1D1-Sepharose matrix was separated from the unbound fraction. Proteins were resolved on a 9% SDS-polyacrylamide gel and stained with Coomassie Blue and subjected to autoradiography for analysis of phosphorylated proteins. lane a, unbound fraction for ROS membranes phosphorylated in HEPES; lane b, unbound fraction for ROS phosphorylated in the presence of ROS soluble fraction; lane c, bound fraction containing the channel (α- and β-subunits) for ROS phosphorylated in HEPES; lane d, bound fraction containing the channel (α- and β-subunits) for ROS phosphorylated in the presence of ROS soluble fraction. The β-subunit is most intensely labeled when phosphorylation is carried out in the presence of ROS soluble fraction.