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Fig. 2. High efficiency of Cre-mediated plectin deletion. (A) Immunoblotting of epidermal and dermal tissue homogenates from newborn Plec^flox/flox (control) and K5-Cre KO mice. After separating epidermis and dermis using dispase, proteins were extracted and subjected to immunoblotting using anti-pan-plectin antiserum. Tubulin served as loading control. Note, very faint plectin signal in K5-Cre KO epidermis, testifying to nearly complete abolition of plectin in this tissue, whereas in dermis, plectin expression was hardly reduced. (B) Southern blot analysis of genomic DNAs from epidermis and dermis of newborn K5-Cre KO mice and their Plec^flox/flox (control) littermates. DNAs were digested with NcoI (releasing 15 kb and 9 kb fragments from the Plec^flox and Plec alleles, respectively) and hybridized with the Nco probe (see Fig. S1A in supplementary material; Plec^flox and Plec alleles, respectively). Note complete conversion of the Plec^flox to the Plec allele in the epidermis of K5-Cre KO mice and the presence of some Cre activity in the dermis, as indicated by the 9 kb Plec allele. (C) Immunofluorescence microscopy of frozen skin (a,b), tongue and palate (c,d), esophagus (e,f) and heart (g,h) tissue sections of newborn control and K5-Cre KO specimens using anti-pan-plectin antiserum. Note strong plectin signal in control skin along the basal membrane (arrowheads in a) and throughout the epidermis. In the oral cavity, the plectin signal is most prominent along the basal membranes of palate and tongue epithelia (arrowheads in c). Plectin was also expressed in the dermis, foremost at dermo-epidermal junctions of hair follicles (asterisks in a). No plectin-specific label was detectable in K5-Cre KO epidermis, palate, and tongue epithelia, except for a few single epidermal cells, most likely melanocytes and Langerhans cells (arrows in b). Also note strongly reduced plectin-specific label in K5-Cre KO esophagus (f), but in K5-Cre KO heart, plectin expression was not reduced (h). p, palate; t, tongue. Dashed lines (in b and d) indicate dermo-epidermal borders. Bars, 20 µm.

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