Figure 6.

Fractionation of filamin, RalA, and actin proteins. (A) Cos7 cells were transfected with vector (control), wild-type RalA (WT), V23RalA (V23), or N28RalA (N28). Cells were serum-starved and Triton-solubilized, and insoluble cytoskeletal fractions were prepared after cell lysis. Samples of each fraction were immunoblotted with antifilamin antibody. The relative amounts of filamin in the cytoskeletons were quantitated from digitized images of autoradiograms of immunoblots by using a National Institutes of Health image analysis program. Total filamin protein represents the sum of the amounts of filamin in Triton-soluble and insoluble fractions. Each value represents the percentage of total and the mean ± SE (n = 5). Statistical significance was determined by the one-factor ANOVA (P < 0.05). (Inset) The representative autoradiograph of the cytoskeleton-associated filamin in cells transfected with control (a), wild-type RalA (b), V23RalA (c), and N28RalA (d) probed with antifilamin antibody. (B) Samples of Triton-soluble and insoluble fractions from RalA transfectants were immunoblotted with anti-RalA antibody. The relative amounts of RalA proteins were analyzed as in A and are presented as filled bars. The relative content of actin was determined by scanning of Coomassie brilliant blue-stained gel and National Institutes of Health image analysis program and are presented as open bars. Each value represents the mean ± SE (n = 4).