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. 2004 Sep 13;166(6):901–912. doi: 10.1083/jcb.200404024

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Copyright © 2004, The Rockefeller University Press

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Figure 6. — Actopaxin phosphorylation mimics Rac activity. WT, Quint, and S4/8D clones plated on glass coverslips were cotransfected with dominant negative N17 Rac (DN Rac) and GFP for 24 h. (A) After transfection, slips were fixed and processed for indirect immunofluorescence. Actin stress fibers were visualized by staining with rhodamine phalloidin. Bar, 5 μm. (B) Quantitation of lamellipodia formation in WT, Quint, and S4/8D transfected cells cotransfected with DN Rac was accomplished by counting GFP positive cells and compared with control nontransfected cells. The presence of at least one broad lamellipodia on a cell was counted as positive. Note that cells expressing the S4/8D actopaxin mutant were more resistant to inhibition of lamellipodia formation than the WT cells suggesting phosphorylation of actopaxin is downstream of Rac. Percentages are representative of three separate experiments and a total of 300 cells.