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Fig. 4. Inducible knockout of plectin in mice and examination of epidermal integrity. (A) Southern blot analysis (autoradiography) of DNA isolated from the epidermis of a PLECflox/–:K5-Cre-ERT mouse after treatment with 4-hydroxy-tamoxifen (OHT). DNA was digested with NcoI as described in Fig. 2, and hybridized with the Nco probe shown in Fig. S1A. The 2.8 kb signal stems from the plectin-null allele. Note partial Cre-mediated recombination after OHT treatment. (B-D) Immunofluorescence microscopy of frozen leg (B), ear (C) and tail (D) skin sections of OHT-treated PLECflox/–:K5-Cre-ERT mouse specimens using anti-pan-plectin antiserum. Note patches of plectin-deficient keratinocytes in intrafollicular epidermis (brackets). Arrows in B indicate non-keratinocyte plectin-positive cells, most likely melanocytes and Langerhans cells. HF, hair follicle. SG, sebaceous gland. (E-G) H/E stainings of tail skin (E,F) and shaved back skin (G) specimens from OHT-treated PLECflox/–:K5-Cre-ERT mice, showing normal skin morphology and absence of microblisters in unstressed skin (E) and formation of microblisters (asterisks) after repeated (10x) tape strippings with D-Squame disks (F,G). (H,I) Transepidermal water loss (TEWL; H) and quantification of stratum corneum protein removal (I) after serial tape strippings of skins from Plecflox/–:K5-Cre-ERT mice, that had been either untreated (–OHT) or treated (+OHT). Basal TEWL was measured before the first stripping. Values are mean ± s.e.m. from 5 OHT-treated and 5 untreated (control) Plecflox/–:K5-Cre-ERT littermates. Measurements were taken after two subsequent tape strippings. For statistical analyses the Student's t-test was used (*P<0.1; **P<0.05). Note that for quantification of stratum corneum protein removal, disks from two subsequent strippings were pooled. Bars, 20 µm.
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