FIG. 2.

mDia1 deficiency impairs formation of actin fibers and peripheral targeting of v-Src upon temperature shift. (A) Time course of formation of actin fibers and v-Src translocation after temperature shift. WT-NY72 and KO-NY72 cells grown at the nonpermissive temperature were shifted to the permissive temperature and maintained for 0, 1, 4, and 24 h. The cells were fixed and stained with an antibody against Src and Texas Red X-phalloidin. A merged image of the two populations of cells 4 h after the temperature shift is shown in the figure in the supplemental material. Bar, 50 μm. (B) Quantification of F-actin intensity. WT-NY72 and KO-NY72 cells were cultured for 0, 1, 4, and 24 h after the temperature shift and stained for F-actin with Texas Red X-phalloidin. The fluorescence density, i.e., the intensity divided by cell area, of each cell was quantified and is shown (n = 20 each). (C) Accumulation of v-Src in the P100 fraction. WT-NY72 and KO-NY72 cells were shifted to the permissive temperature and harvested at indicated times. The P100 fraction was prepared from each population and subjected to Western blotting for Src. v-Src signal was detected 5 kDa below the molecular mass of c-Src. The c-Src signals were not visible under the present blotting conditions. The density of each band is shown below the blot.