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Fig. 1. Colocalisation of fascin and PKC
is regulated by PKC and Rac activity. (A,B) SW480-Pa cells plated on 15 nM LN for 4 hours in the absence or presence of 1 µM BIM or 100 µM NSC23766 were co-stained with antibodies as stated, and imaged using phase-contrast or confocal microscopy. Scale bars: 10 µm. Insets show higher magnification views of the edges of representative control cells; scale bars: 4 µm. In B, arrows and arrowhead indicate cell edges positive or negative for Rac, respectively. (C,D) PKC activation is Rac dependent. SW480-Pa cells were pre-treated with BIM, TPA, NSC23766 or solvent (Con) as described in the Materials and Methods, lysed and analysed by SDS-PAGE and immunoblotting for total PKC or PKC–T655PO4 as a reporter of PKC activation (C). D shows quantification of results from three independent experiments. Columns represent the mean and bars indicate s.e.m. (E) PKC was immunoprecipitated from LN-adherent cells and treated in vitro with 100 µM NSC23766 or 1 µM BIM before activity analysis by immunoblotting. Only BIM treatment decreased PKC–T655PO4. Representative of three independent experiments.
