Figure 5.

(A) In split CFTR, tightly bound [α³²P]8-azido nucleotide predominantly labels NBD1. (Left) Autoradiogram from membranes of oocytes coexpressing Flag-3–835 and Flag-837–1480 (+), or of noninjected oocytes (−) incubated for 15 min at 30°C with 5 μM [α³²P]8-N₃ATP and 10 mM Mg²⁺ ± 1 mM V_i, subjected to the standard wash procedure including 5-min postincubation at 30°C, irradiated with UV, immunoprecipitated, and subjected to SDS-PAGE. Arrow indicates photolabeled band corresponding to CFTR half containing NBD1. (Right) Proteins from the same membranes were immunoprecipitated and blotted with either anti–R-domain antibody, which recognizes the half of CFTR containing NBD1 (left two lanes), or with anti–COOH-terminal antibody, which recognizes the half containing NBD2 (right two lanes). (B) Extremely slow release of [α³²P]8-azido nucleotide during nucleotide-free postincubation at 30°C before UV irradiation. Initial incubation of membranes from HEK293T cells expressing Flag-CFTR was with 5 μM [α³²P]8-N₃ATP for 15 min at 30°C with (+V_i) or without (−V_i) 1 mM V_i. After postincubation for time indicated, immunoprecipitation, and SDS-PAGE, the resulting autoradiogram signals were normalized to that obtained with V_i but without postincubation (postincubation time = 0) in each experiment (n = 6 for 0- and 5-min points; n = 2 for 1.5- and 15-min points; error bars give ± SD). Curves show simultaneous least-squares fits to both datasets with single time constant, yielding τ = 15 ± 2 min.