Figure 6.

Endogenous PKD1 remains associated to the plasma membrane in the presence of inhibitors of the group I PAK or in cells transfected with siRNAs targeting PAK1 and PAK2. Confluent cultures of IEC-18 cells were treated in the absence (A) or presence of 3.5 μm PF-3758309 (B), 5 μm FRAX597 (C), or 30 μm IPA-3 (D) for 1 h and subsequently stimulated with 10 nm ANG II for various times, as indicated. E and F, cultures of IEC-18 cells were transfected with non-targeting siRNA (Non. Targ) or with a mixture of siRNAs targeting PAK1 and PAK2 (siPAK1+2) for 5 days. Then, the cultures were stimulated with 10 nm ANGII for the indicated times. The cultures were then washed and fixed with 4% paraformaldehyde and stained with an antibody that detects the PKD1 and Hoechst 33342 stain to visualize the nuclei. The bars represent the analysis of the relative change in plasma membrane fluorescence intensity (expressed as ratio of membrane/cytosol).