Figure 3.
14-3-3 Binding Inhibits Rnd3 by Inducing Translocation from the Plasma Membrane to the Cytosol
(A and B) NIH 3T3 cells were cotransfected with the indicated constructs. (A) Cell morphology quantification. Graph shows pooled results from technical triplicates in three independent experiments (n = 300 cells/condition/experiment). Conditions with the same number (1, 2, 3) are not statistically significantly different from each other. Error bars indicate mean ± SEM; Table S2 shows statistical analysis between the conditions. (B) Single confocal images (maximum intensity z stack projections in merge) showing cell morphology and FLAG-Rnd3 localization. Arrows: FLAG-Rnd3-expressing cells (F-actin images) with a rounded phenotype with protrusions. Arrowheads: normal phenotype. Scale bar, 20 μm.
(C) The indicated constructs were transfected into COS7 cells prior to cell lysis and biochemical fractionation. Fractions were immunoblotted with indicated antibodies. ERK1/2 and transferrin receptor are markers of cytosolic and membrane fractions, respectively. As controls (bottom panels), cells were transfected with the Rnd3 nonisoprenylated mutant S241 or Rnd3 WT with staurosporine (stau.), PMA, or calyculin A (cal. A) treatment. β, 14-3-3β; ROCK1, myc-ROCK11-420; T, total extracts; N, nuclear fraction; C, cytosolic fraction; M, membrane fraction. See also Figure S3 and Table S2.