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Fig. 5. Knock down of epiplakin by RNAi. (A) Immunostaining of HeLa cells transiently transfected with siRNA-SC (a) and siRNA-KD (b). Cells were fixed and stained with the anti-epiplakin (epip) antibody. Nuclei were stained with TO-PRO 3 (blue). Bars, 30 µm. (B) Western blotting analysis of epiplakin expression in HeLa cells transiently transfected with siRNA-SC or siRNA-KD (2, 5, 10, 25, and 50 nM). Cellular extracts were fractionated on a 3% SDS-NuPAGE gel and probed with anti-epiplakin antibody. ß-actin is shown as a loading control. The level of epiplakin was measured by densitometry and normalized to ß-actin in each corresponding sample. The data (mean ± s.d.) are averages from three separate experiments with duplicate samples and are presented as a fold change from the sample of siRNA-KD over that of siRNA-SC. (C) Immunostaining analysis of HeLa cells transiently transfected with siRNA-KD. (a-d) Cells were subjected to triple-label staining with the antibodies specific for epiplakin, vimentin and keratin. (e,f) In a parallel experiment, cells were double-stained with antibodies for keratin (kerat) and ß-actin (actin). The non-transfected cells are indicated by arrows (a-f). Bars, 20 µm.
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