FIG.5.

Function and localization of wild-type UNC-73B and PH domain mutants in a cell culture-based assay. (A) Effects of UNC-73B mutants on actin polymerization in Rat2 fibroblasts. Rat2 fibroblasts were injected into the nucleus with plasmid DNA encoding wild-type GFP/UNC-73B DH/PH/SH3 (panels i and i*); the ΔPH (panels ii and ii*), W1502A (panels iii and iii*), KR-to-AA (panels iv and iv*), and KR-to-EE (panels v and v*) mutants; and Flag-V12 Rac (panels vi and vii*). Three hours after injection, cells were fixed and subsequently stained with anti-GFP (Abcam Ab290) or M2 (anti-Flag) antibody for identification of injected cells and filamentous actin (Texas Red phalloidin). Actin staining is present in the initial panel for each injected construct, while the anti-GFP or anti-Flag staining is present in the asterisk-labeled panels, as appropriate. (B) Localization of ΔPH and W1502A in Rac-activated cells. GFP constructs of the two mutants of UNC-73B PH domain (ΔPH and W1502A) were coinjected in the nuclei of Rat2 cells with Flag-V12 Rac in the presence of serum to induce membrane ruffling. Cells were fixed and stained for actin and anti-GFP (α-GFP) as for panel A. Actin and anti-GFP staining, as well as the merged images of the two, are shown.