Nef induces CFL1 phosphorylation. A) 293 cells were transfected with Nef/Stop, Nef/WT or Nef/G2A plasmids for 36 h. Cell lysates were then subject to Western blotting using antibodies to Nef (1:2,000), total CFL1 (1:1,000), phospho-serine-3-CFL1 (1:2,000), and GAPDH (1:5,000). The intensity of the phospho-serine-3-CFL1 bands was quantified and normalized to Nef/Stop that was assigned a value of 1.0. B and C) 293 cells transfected with either Flag-tagged Nef/WT or Flag-tagged Nef/G2A plasmids were fixed, permeabilized, and blocked with BSA (as described in Experimental Procedures), incubated with anti-Flag (B) or anti-Flag and anti-phospho-serine-3-CFL1 antibodies (C) (1:500 in 5% BSA/PBS) for 2 h, and stained with FITC-conjugated (B) or FITC-conjugated and Alex-Fluor-555 labeled secondary antibodies (C). The cells were mounted in Prolong Gold Antifade Reagent before immunofluorescence microscopy. Fig 5B also shows the phase contrast image of 293 cells expressing Flag-tagged Nef/WT exhibiting "rounding off" phenotype unlike cells expressing Flag-tagged Nef/G2A. The arrows in Fig. 5B identify non-Nef expressing cells. The panels in Fig 5C are for Nef, p-CFL1, and merged images. The arrows in Fig. 5C identify Nef expressing cells. Bar, 5 um. D) The densitometric mean intensity of p-CFL1 stained cells was quantified from Nef/WT and Nef/G2A transfected cells and compared to non-Nef expressing cells.