Fig. 3. Up-regulation of epiplakin expression in keratinocytes. (A) Immunofluorescent staining of NHEK cells maintained in low calcium (a) and high calcium for 1 day (b) or high calcium for 3 days (c), fixed and stained with anti-epiplakin (red) antibody and TO-PRO 3 for nuclei (blue). Bars, 20 µm. (B,C) Immunoblotting of soluble (B) and insoluble (C) fractions from NHEK-L (lane 2) or NHEK-H of 1 (lane 3), 3 (lane 4), 5 (lane 5), 7 (lane 6) and 9 days (lane 7) were probed with epiplakin antibody. ß-tubulin is shown as a loading control. (D) Real-time RT-PCR analysis of epiplakin and involucrin transcripts in NHEK cells. The threshold values (Ct) of epiplakin and involucrin were normalized by the subtraction of the Ct value of ß-actin in each corresponding samples (insert: epiplakin, black line; involucrin, blue line). Three separate experiments were performed with triplicate samples. The data are presented as a fold change (mean ± s.d.) of the relative level of epiplakin (black and white bars) and involucrin (hatched bar) from NHEK-H or HeLa over that of NHEK-L sample.