Mapping of the FlnA-PACSIN2 interface. (A) GST-PACSIN2 F-BAR domain was incubated with His-tagged FlnA recombinant truncates as indicated. Complexes were pulled down with glutathione Sepharose beads, subjected to SDS-PAGE, and probed for His (FlnA). FlnA rod 2 (repeats 16-23; 83 kDa) and repeat 20 (44 kDa) were collected with glutathione Sepharose beads. FlnA domains are shown on the top of the panel. The arrowhead points to repeat 20. (B) Electron microscopy of FlnA and GST-PACSIN2 F-BAR domain (P2) observed by rotary shadowing. PACSIN2 binds ∼15 nm from the FlnA self-association site. Arrowheads point to PACSIN2 bound along FlnA molecules. (C) FlnA-binding motif of PACSIN2 compared with that of known FlnA partners. Protein names, amino acid sequences, and positions are indicated. Nonpolar residues are colored green, neutral are magenta, basic are blue, and acidic are red. *Nonpolar or neutral residue. The arrowhead points to PACSIN2 Pro180. (D) GST-PACSIN2 F-BAR domain WT or P180A was incubated with His-FlnA repeats 16 to 23. Complexes were pulled down with glutathione Sepharose beads, subjected to SDS-PAGE, and probed for His (FlnA).