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. 1997 Dec 15;139(6):1455–1464. doi: 10.1083/jcb.139.6.1455

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Ability of a mutant receptor, s47WA, to (A) bind to API and (B) induce liposome binding. (A) Indicated amounts of biotinylated s47 or s47WA protein were incubated with equal amounts of PI-PLC–released API protein in a total volume of 200 μl for 1 h on ice. Samples were immunoprecipitated with the anti-DAF antibody, separated by SDS-15% PAGE and transferred to nitrocellulose. Blots were probed with streptavidin-HRP and visualized by enhanced chemiluminescence. 1× = 0.36 μg protein. (B) Biotinylated PI-PLC–released API was incubated with 0.36 μg (1×) s47 or 3.6 μg (10×), 36 μg (100×), or 90 μg (250×) s47WA protein at 4°C for 20 min. Liposomes were added and samples were incubated at 37°C for 5 min before centrifugation. Fractions were analyzed as described in the legend to Fig. 1. Similar results were obtained with the s47WA mutant (100×) when the 4°C incubation step was increased to 90 min.