Fig. 7. Assessment of Rho activity and intercellular junctions in keratinocytes with kazrin knockdown. (A) Relative levels of kazrin mRNA as determined by quantitative PCR in keratinocytes infected with kazrin siRNA constructs 426 or 1077 or the scrambled (scr) control. Bar chart shows the average values from three independent infections (means ± s.d.). (B) Immunoblotting of protein extracts from keratinocytes expressing kazrin siRNA426, siRNA1077 or the scrambled control. Band intensities were quantified and kazrin levels, relative to the scrambled controls and normalized to actin, are indicated below immunoblots. (C) Pulldown using GST-Rhotekin Rho-binding domain coupled to glutathione agarose and lysates from keratinocytes stably expressing siRNA426, siRNA1077 or the scrambled control. 5% of each lysate used for the pulldown assays was probed with antibodies to Rho (Rho total), kazrin or actin. (D) Keratinocytes expressing siRNA426 or the scrambled control were transferred to high Ca²⁺ medium for 2 hours prior to fixation and staining for F-actin (phalloidin) or desmoplakin. Bar, 50 µm. (E) Quantification of average pixel intensity of desmoplakin immunostaining at cell-cell borders of keratinocytes expressing siRNA426 or the scrambled control. Data are the means ± s.d. of three independent experiments.