Nef induces LIMK1 activation. 293 cells were transfected with either Flag-tagged Nef/WT (A) or Flag-tagged Nef/G2A (B) plasmids. After 36 h cells were fixed, permeabilized, and blocked with BSA as described in Experimental Procedures. Cells were incubated with anti-Flag tag (1:500 in 5% BSA/PBS) and phospho-specific LIMK1 (1:250 in 5% BSA/PBS) antibodies for 2 h and labeled with FITC-conjugated (Flag) and Alex-Fluor-555 labeled (phospho-CFL1) secondary antibodies. The cells were mounted in Prolong Gold Antifade Reagent before immunofluorescence microscopy. The panels in the figure are for Nef, p-LIMK1, and merged images. Arrows identify Nef expressing cells. Bar, 5 um. B) The densitometric mean intensity of p-LIMK1 stained cells was quantified from Nef/WT and Nef/G2A transfected cells and compared to non-Nef expressing cells.