Fig. 4.

RACK1 interacts with FlnA through its WD4 and WD6 repeats. Peptides encoding individual WD repeats of RACK1 were tested for their ability to compete with full-length RACK1 for binding to FlnA. A: structure of human RACK1 (50). Colored areas show the location of each WD peptide, corresponding to the equivalently colored residues in the sequence. B: immunopurified FlnA was incubated with WD peptides and overlaid onto immobilized RACK1 using a slot-blot filter apparatus, the membrane was probed with anti-FlnA antibody and FlnA binding was quantitated by densitometry. Representative binding experiment; the first 2 lanes contain no immobilized RACK1, showing negligible nonspecific binding of FlnA. Preincubation of FlnA with peptides from WD4 and WD6 repeats reduces FlnA binding to RACK1. C: quantitation of FlnA binding to RACK1 in the presence of WD repeat peptides (means ± SE from 9 experiments). WD4- and WD6-peptides compete with full-length RACK1, causing significant (*P < 0.05; **P < 0.01) reductions in bound FlnA. D: concentration-dependent reduction of FlnA binding to RACK1 after preincubation with WD4- or WD6-peptides, showing that the peptides specifically compete for FlnA.