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Fig. 6. Transfection with a constitutively active PKC mutant increases ezrin T567 phosphorylation in CACO-2 cells. CACO-2 cells (A-H) were transfected under the same conditions described in Fig. 5 with a V5-tagged A120E PKC mutant, on day 4 and fixed on day 5 (A-D), or transfected on day 2 and fixed on day 3 (E-H) of culture. CACO-2 C2Bbe cells transduced with a lentivirus expressing the shRNA anti-PKC and selected in puromycin, were transfected with the same mutant on day 4 and fixed on day 5 after plating (I-M). The cells were processed with anti-T567-P ezrin antibody (green) and anti-V5 tag antibody (red), and counterstained with DAPI (blue). The images shown in a X-Y confocal plane at the apical level (A,B,E), at the basal level (F) or in X-Z 3D reconstructions (C,D,G,H,L,M). I-K are projections of the entire confocal stack separated in the individual channels. The red channel was omitted in A,C,H,J,M to show T567-P signal alone. Arrows indicate transfected cells. Arrowheads point to the basal surface in X-Z reconstructions. Scale bars: 10 µm.

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