FIGURE 3:

Identification of two binding sites for the dAbp1 SH3 domain in PakB. (A) The N-terminal region of PakB contains seven PxxP motifs (P1–P7; red underlined). (B) Pull-down assays were performed using GST fusion proteins containing the SH3 domains of dAbp1, MyoB, or MyoC and lysates of PakBˉ cells expressing the indicated GFP-PakB constructs. PakB-1-120 containing both the P64V and P86 mutations is designated PakB-1-120-ΔP. The cell lysates and washed pellets were subjected to immunoblot analysis using an anti-GFP antibody. GST-SH3 domains were visualized by Coomassie blue staining. (C) dAbp1 contains a proline-rich sequence similar to the P2–P3/4 region of PakB. (D) Pull-down assays were performed using GST (GST) or GST-dAbp1-SH3 (GST-SH3) and His-tagged dAbp1(His-dAbp1; left) or residues 237–313 of dAbp1 (His-237-313; right). The washed pellets were visualized by Coomassie blue staining after SDS–PAGE.