Fig. 5. KazrinA reduces Rho activity. (A-D) Single confocal slices of keratinocytes transiently transfected with GFP (green; A,B) or HA-kazrinA (green; C,D) in the absence (A,C) or presence (B,D) of Y27632. F-actin was visualized with phalloidin (red) and nuclei with DAPI (blue). Bar, 200 µm. Numbers correspond to shape factors. (E-G) Rho activity is reduced in keratinocytes infected with HA-kazrinA whereas Rac activity is unaffected. (E,F) Pulldown assays using GST-Rhotekin Rho-binding domain (E) or GST-PAK p21 binding domain (F) coupled to glutathione agarose in keratinocyte lysates infected with GFP or HA-kazrinA (active). 5% of each lysate used for the pulldown assays was probed with antibodies to Rho or Rac1 (total), kazrinA (KA), β-tubulin or (β-tub) GAPDH. (G) Quantification of three independent Rho pulldown experiments from different infections. Percentage active Rho was calculated by dividing the band intensity of Rho active with the band intensity of Rho total. Values are expressed relative to percentage of active Rho in GFP-infected cells as 100%. Data are the mean ± s.d. Statistical significance was calculated using the paired Student's t-test (^*P<0.05, n=3). (H-L) Rescue of kazrinA-induced changes in shape and actin cytoskeleton by dominant active V14RhoA. (H) Keratinocytes co-expressing kazrinA with GFP or V14RhoA were scored as `rescued' if they had normal morphology and a cortical actin band. At least 100 cells were counted per transfection in each experiment. Data are the means ± s.d. of three experiments. Statistical significance relative to GFP control was assessed by the paired Student's t-test (^**P0.0005; n=3). (I-L) Composite images of z-stacks showing keratinocytes co-transfected with HA-kazrinA (red) and either GFP (green; I,J) or myc-tagged V14RhoA (green; K,L). F-actin was visualized with phalloidin (blue). Open arrows indicate cells expressing kazrinA alone or together with GFP. Filled arrow indicates cell solely expressing V14RhoA and arrowhead indicates cell expressing both kazrinA and V14RhoA. Bars, 60 µm. (M-P) Desmoplakin levels are increased at cell-cell borders of kazrinA and V14RhoA co-transfectants, relative to cells expressing kazrinA alone. (M) Quantification of average pixel intensity of desmoplakin at cell-cell borders of cells expressing kazrinA alone or in combination with V14RhoA. Data are the means ± s.d. of three independent experiments. Statistical significance was determined with paired Student's t-test (^*P<0.05; n=3). (N-P) Desmoplakin (green) levels at borders of cell solely expressing kazrinA (blue; N) are lower than those in cells co-expressing V14RhoA (red; O,P). Images are composites of z-stacks. Bar, 7.5 µm.