FIGURE 3.

JRAB directly binds to F-actin, induces cross-linking, and inhibits depolymerization of F-actin. A, panel a, each recombinant protein was incubated with F-actin and centrifuged at 125,000 × g. An aliquot of the pellet (P) and supernatant (S) was subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining. Top panels, presence of F-actin; bottom panels, absence of F-actin. Asterisks, actin; arrowheads, recombinant proteins. Panel b, graph shows the ratio of each recombinant protein in the pellet versus total recombinant protein in the pellet and supernatant (P/(P + S)). Black bar, the negative control (GST); open bars, the proteins with F-actin-binding activity; gray bars, the proteins with no significant F-actin-binding activity. Panel c, schema indicates the ability of each protein to bind to F-actin. B, F-actin cross-linking activity of JRAB. Panel a, indicated recombinant proteins were incubated with F-actin and centrifuged at 16,000 × g to separate the pellet (P) and supernatant (S). Asterisks, actin; arrowheads, recombinant proteins. Panel b, graph shows the ratio of each recombinant protein in the pellet versus total recombinant protein in the pellet and supernatant (P/(P + S)). Black bar, the negative control (GST); open bars, the proteins with F-actin cross-linking activity; gray bars, the proteins with no significant cross-linking activity. Panel c, schema indicates the ability of each protein to cross-link F-actin. C, effect of JRAB on actin depolymerization. Panels a and b, indicated recombinant proteins were incubated with pyrene-labeled F-actin diluted to the critical concentration, and fluorescence was monitored using a spectrofluorometer. The results shown are representative of at least three independent experiments.