Actin_F352A suppresses the cell rounding phenotype caused by RavK.

A. Actin_F352A but not wild-type actin suppressed the cell rounding phenotype caused by RavK. HEK293T cells were transfected with (i) pEgfp-ravK alone, (ii) pEgfp-ravK together with pCMV-Flag-actin or (iii) pEgfp-ravK together with pCMV-Flag-actin_F352A for 24 h, and were observed under a fluorescence microscope. Bar, 50 μm. B. Expression of GFP-RavK, Flag-actin or Flag-actin_F352A. Cells were transfected by indicated plasmids for 24 h, and the cleared lysates from transfected samples were resolved by SDS/PAGE, and subjected to immunoblotting with antibodies specific to GFP and the Flag tag, respectively. α-tubulin was used as a loading control. C. Quantification of the percentage of green cells exhibiting the cell rounding phenotype. Experiments were performed in triplicate and at least 200 cells were examined in each sample. Error bars indicate standard error of the mean (SEM); N.S., not significant; ****, p<0.0001. Similar results were obtained in three independent experiments.