Fig. 3. Periplakin expression in MCF-7 monolayers and wound edges. (A-D) Intact monolayers were stained with (A) Desmoplakin and periplakin (red and green channels and merged image); XZ-projection confirms partial co-localisation the two plakins at cell borders. Note also the distinct cytoplasmic pool of periplakin, (B) Keratin 8 and Periplakin, (C) Periplakin and ß-catenin, (D) Actin and periplakin. Note that cytoplasmic periplakin is not co-localised with either keratin or actin networks in monolayer cells. (E-G) Wound edge cells stained with (E) Periplakin (left panel at both time points) and desmoplakin (right at both time points) 30 minutes and 2 hours after wounding. Desmoplakin, unlike periplakin is retained at the free wound edge at the 30 minutes time point. (F) Periplakin (green) and ß-catenin (red) merged image, 30 minutes after wounding. (G) Periplakin (red) and Actin (green), 2 hours after wounding. (H) western blot of the subcellular distribution of periplakin (Ppl), Desmoplakin (Dpl) and Keratin 18 (K18) in Saponin-soluble (S1 pool), Triton X-100 soluble (S2 pool) and insoluble pools (P3). Quantification of the relative distribution of periplakin and desmoplakin in the fractions was performed by densitometry of four independent fractionation experiments. (I) Immunofluorescence staining of cell extracted on coverslips confirming that Triton-insoluble periplakin (left panel) and desmoplakin (right panel) are located at cell borders.