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Fig. 7. Dependence of NO effects on PKG I and PKG II and guanylate cyclase. (a,c) Distribution of WT GFP-VASP in human PTEC co-transfected with siRNA targeting (a) PKG I or (c) PKG II (supplementary material Movies 5 or 6, respectively). Montage of cell images taken immediately before (left panels) and after (right panels) addition of NO donor, showing distribution of WT GFP-VASP. Time points at which images were taken are shown in each panel. White lines in each image show the axises used to generate the kymographs shown in b and d. Bars, 10 µm. (b and d) Kymographs of the cell in panels a and c, respectively, using the axis as shown. Arrows indicate the time at which the NO donor was added. (e,f) Localization of WT GFP-VASP within lamellipodia of cells co-transfected with siRNA targeting (e) PKG I or (f) PKG II, measured as a percentage of the value immediately before addition of NO donor (control=100%). Values are the mean ± s.e.m. at the indicated times after addition of NO donor; n=8 (PKG I) and n=10 (PKG II) separate determinations. The change in percentage of WT GFP-VASP within lamellipodia in response to NO donor addition was significant for cells transfected with siRNA targeting PKG I (P<0.0001, ANOVA) but not for siRNA targeting PKG II (P=0.8841, ANOVA). (g) Effect of the siRNA on PKGI or PKG II protein levels in cells transfected with control siRNA (C) or siRNAs specifically targeting the indicated PKG isoforms (+). Loading control (actin) is shown in the lower panel. (h) Change in cell area following addition of NO donor to cells transfected with siRNA targeting PKG I (black bars) or PKG II (white bars). Values were determined as described for Fig. 4b. Results are given as the mean ± s.e.m.; n=13 (PKG I) and n=11 (PKG II) individual determinations. The difference between the area after addition of NO donor in PKG I transfected cells was significantly reduced by NO addition (***P<0.01, two-tailed t-test). (i) Localization of WT GFP-VASP within lamellipodia of cells treated with ODQ, measured as a percentage of the value immediately before addition of NO donor (control=100%). Addition of ODQ for 30 minutes produced a small and non-significant decline in WT GFP-VASP within lamellipodia. Values are the mean ± s.e.m. at the indicated times after addition of NO donor of six separate determinations. The change in percentage of WT GFP-VASP within lamellipodia in response to NO donor was not significant for cells treated with ODQ (P>0.05, ANOVA). (j) Change in cell area following addition of NO donor to cells treated with ODQ. Values were determined as described for Fig. 4b. Results are mean ± s.e.m.; n=6. The difference between the area after NO addition in ODQ treated cells was not significant (P>0.05, two-tailed t-test).
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