Fig. 5. Fhos forms a homotypic complex via the FH2 domain. (A) HeLa cells co-expressing Myc-tagged Fhos-F and GFP-fused Fhos mutants were lysed, and proteins were immunoprecipitated with the anti-Myc antibody. The precipitants were analyzed by immunoblot with the anti-GFP antibodies. Proteins in cell lysates were also analyzed directly by immunoblot (lower panel). (B) HeLa cells co-expressing Flag-tagged Fhos-FH2 and Myc-tagged Fhos mutants (FH1FH2 or N) were lysed and immunoprecipitated with the anti-Flag antibody. The immunoprecipitates were analyzed by immunoblot with the anti-Myc or anti-Flag antibodies. Proteins in cell lysates were also analyzed directly by immunoblot. (C) HeLa cells co-expressing Myc-tagged Fhos-FH2 and GFP-fused Fhos-NC. Cells were fixed and detected by triple-fluorescence microscopy for GFP fluorescence, Myc immunostaining and phalloidin staining. Merged images are shown in the lowest panel. (D) The direct interaction of MBP-FH2 with GST-FH2. MBP-FH2 or MBP alone was incubated with GST-FH2 or GST-DAD. Proteins were pulled down with glutathione-Sepharose-4B and subjected to SDS-PAGE, and stained with CBB. (E) The direct interaction between His-tagged FH1FH2 and GST-FH2. His-FH1FH2 was incubated with GSTFH2 or GST alone. An in vitro pull-down binding assay was performed as in Fig. 5D.

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