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Fig. 5. Wound closure and keratin organisation in stable cell lines expressing PPL C-terminus. (A) Immunoprecipitation and western blot of Periplakin C-terminal linker domain by HA-tag antibody. MCF, untransfected parental cells; E, empty vector transfected clone, C-ter, representative clone of the linker domain transfected cells. Right panel: western blotting of endogenous periplakin and C-terminal domain with TD2 antibody. (B) Wound closure in control and Ppl-C cell lines. Wounded monolayers grown on glass coverslips were stained with FITC-phalloidin to visualise the wound edges. (C) Quantification of the wound closure in empty vector and PPL C-terminus transfected cell clones. Confluent monolayers grown in 6-well plates were wounded with pipette tip. The wound closure was followed by photographing phase contrast microscope fields after 15 minutes, 8 and 24 hours. For each wound the width of the wound after 15 minutes was designated as 100% and the subsequent time-points in the graph show the relative width of the open wound. Mean and standard deviation of ten measurements are shown. By the 24 hours time-point the control wounds were completely closed. (D). Keratin 8 immunofluorescence at wound edges. Panel (a) empty vector transfected control cell line. Panels (b) and (c) Keratin immunofluorescence at the edges of two independent wounds of a clone expressing periplakin C-terminus. (E) Ser431-phosphorylated keratin expression at wound edges. Panel (a) control; panel (b) cell line expressing Ppl C-terminus. (F) western blotting of Ser-431 phosphorylated keratin in monolayers (m) and wounded (w, ten wounds per 100 mm cell culture dish) control and Ppl C-terminal cell clones. Keratin 18 blot of the same membrane shows that there is no difference in the total keratin expression between the cell clones or treatments. Scale bar is 100 µm in B, C, E and F and 20 µm in G-K.
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