Fig. 4. Localization of active Fhos to actin stress fibers is mediated via its N-terminal F-actin-binding region. (A) Binding of Fhos to F-actin in a cosedimentation assay. Polymerized F-actin and His-tagged Fhos-N (1-569) were mixed in F-buffer to a final concentration of 6.6 µM and 1.3 µM, respectively, and incubated for 60 minutes at 25°C. The mixture was centrifuged for 60 minutes at 100,000 g, and both supernatants (S) and pellets (P) were analyzed by SDS-PAGE, followed by staining with CBB. (B) Quantitative analysis for binding of Fhos-N to F-actin. Various amounts of His-tagged Fhos-N (1-20 µg) were incubated with 1 µg of polymerized actin in a total volume of 50 µl. After ultracentrifugation, the free and bound His–Fhos-N were subjected to SDS-PAGE followed by staining with CBB. The amounts of the protein on the gel were estimated by an image analyzer. (C) Direct binding of F-actin to GST–Fhos-N in an F-actin overlay assay. Lysates of E. coli expressing GST–Fhos-N, GST–Fhos-FH2 and GST alone were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was probed with F-actin, and bound F-actins were detected using an anti-actin antibody (left panel). Proteins in bacterial lysates were also analyzed by immunoblot with the anti-GST antibodies (middle panel) or CBB staining (right panel). (D) Localization of Myc–Fhos-N to actin stress fibers in HeLa cells. Myc–Fhos-N and Flag–Fhos-FH1FH2 were coexpressed in HeLa cells. The cells were fixed and stained with the anti-Myc antibody (left panel) and phalloidin (right panel). Bars, 20 µm. (E) Direct binding of F-actin to His–Fhos-NC in an F-actin overlay assay. His–Fhos-NC and His–Fhos-FH1FH2 proteins were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was probed with F-actin, and bound F-actins were detected using an anti-actin antibody (left panel). Proteins were also analyzed by immunoblot with the anti-His antibody (right panel).