Fig. 2.

The FH1 phosphorylation suppresses DIAPH1–PFN1 interaction. a HeLa cells were treated with nocodazole for 18 h, and collected by mitotic shake-off. Cell lysates were incubated with GST-PFN1 and then subjected to a GST pull-down assay. The resultant precipitates were subjected to immunoblotting using the indicated antibodies. The DNA content of each sample was determined by FACS. b HeLa cells expressing 3×Flag-DIAPH1-Wt or its FH1 domain were treated and collected as in a. The lysates were incubated with GST-PFN1 and then subjected to a GST pull-down assay. The resultant precipitates were immunoblotted using anti-FLAG antibodies. c, d HeLa cells expressing 3×FLAG-DIAPH1-Wt or the indicated mutants were treated and collected as in a. Cell lysates were subjected to GST-pull-down experiments using GST-PFN1 and immunoblotting using the indicated antibodies. Transferred PVDF membranes were stained with Coomassie Brilliant Blue G-250 (CBB)