Figure 4.—

Pak does not appear to regulate the levels of activated Rho1. (A) Rho activity assay. Using a fusion protein composed of the Rho-binding domain (RBD) of the Rho1-binding protein Rhotekin fused to GST in a GST pull-down assay allowed for the detection of GTP-bound Rho1 in wild-type and pak mutant ovarian tissue lysates. Shown is a representative SDS-PAGE gel Western blotted with anti-Rho1 antibody. The left side shows lanes containing equal volumes of ovarian tissue lysates from wild-type and pak mutant flies. The lanes on the right side show equal volumes of ovarian tissue lysates that were passed through columns of GST-Rhotekin-RBD Sepharose beads and precipitated beads run on a gel. Incubation of these same lanes with anti-GST antibodies revealed amounts of GST-Rhotekin-RBD in each lane. Intensity of Rho1 pull-down bands was normalized against intensity of GST bands and compared to total Rho1 input. (B–E) GFP-based in vivo reporter to detect subcellular changes in activated Rho1 levels. The follicle cell-specific driver tj-Gal4 was used to express UAS-PKNG58AeGFP in a wild-type background (B and C) or a pak mutant background (D and E). Anti-Rho1 antibody shows the level and distribution of total Rho1 whereas anti-GFP antibody shows the level and distribution of activated Rho1. Bar: 50 μm.