FIGURE 5:

The PakB N-terminus binds actin. (A) Time-course images showing the redistribution of PakB-GFP to the cytoplasm after addition of latrunculin A (5 μM) at time 0. Bar, 10 μm. (B, C) Actin filament cosedimentation experiments were performed using (B) GST-PakB-1-120 (1–120) and (C) GST-PakB-1-58 (1–58). Samples were centrifuged at 100,000 × g for 30 min, and the resulting supernatants (S) and pellets (P) were subjected to SDS–PAGE and stained with Coomassie blue (B, left; C) or probed with an anti-GST antibody (B, right). Note that GST-PakB-1-120 and actin have the same mobility on SDS–PAGE. (D) Images of developed migrating PakBˉ cells expressing GFP-PakB-1-58, GFP-PakB-1-79, or GFP-PakB-68-180. Arrows indicate the direction of migration. Bars, 10 μm. (E) Actin filament cosedimentation experiments were performed with GST-dAbp1 in the presence or absence of GST-PakB-1-120 (1–120). Samples were centrifuged at 100,000 × g for 30 min, and the resulting supernatants (S) and pellets (P) were subjected to SDS–PAGE and stained with Coomassie blue. (F) The percentage of GST-dAbp1 that cosedimented with actin in the presence (·) or absence (◯) of 5 μM GST-PakB-1-120 was quantified by densitometry of Coomassie blue–stained SDS gels. Results show the mean and SD for three independent experiments.