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Fig. 3. PKC interacts with ezrin in vivo. Knockdown of PKC with shRNA. (A) CACO-2 cells were transfected with full-length ezrin cDNA (+) or mock transfected (–), and extracted in 0.5% Triton X-100 after 48 hours. Some extracts were immunoprecipitated (ip) with anti-ezrin (+) or with nonimmune IgG (–) as a control. Samples of the extracts (input) and the eluates were probed with anti-PKC antibody (raised in rabbit). The membrane in the middle panel was later reprobed with anti-ezrin antibody (right panel). Mol. mass standards: 85 and 31 kDa. (B-K) CACO-2 C2BBe cells were transduced with nonreplicating lentivirus particles carrying only a gene for puromycin resistance (empty) or an anti-hPKC shRNA sequence under a pol III promoter and the same puromycin-resistance gene (shRNA). (B, C) Confluent monolayers selected in puromycin were extracted in SDS sample buffer at 3, 4, 5, 7 and 10 days of culture. Samples of 30 µg protein were loaded in each lane, run in SDS-PAGE and blotted. The blots were sequentially probed with anti-PKC (B) and then reprobed with anti-tubulin (C) antibodies. Molecular mass standards: 200, 117, 85, 40 and 31 kDa. (D-K) The cells were transduced with empty lentivirus vector (D-G) or with the same vector carrying an anti-PKC shRNA sequence under a pol III promoter (H-K), and selected in puromycin. The cells were fixed and processed with anti-PKC (total protein) antibody (D,F,H,J, green), and with anti-pT555 PKC (active) antibody (E,G,I,K, red), and counterstained with DAPI (blue). D,E,H,I are X-Y projections of the five apical-most confocal sections (2 µm thick) comprising the entire apical region. F,G,J,K are 7-voxel thick X-Z sections of the entire confocal stack corresponding to the image above, shown with the apical side up. Scale bars: 10 µm.

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