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Fig. 7. Establishment of cultured primary keratinocytes from Inv-Dsg2 transgenic and wild-type newborn mouse skin. (A) Immunoblotting analysis for the Flag tag of wild-type and transgenic cell lines grown in low (80 nM; –) or high (1 mM; +) calcium for 5 days. Dsg2-Flag (160 kDa) expression was detected in the calcium-treated transgenic but not the untreated transgenic or the wild-type control keratinocytes. Differentiation was confirmed by increased involucrin expression (*) in response to calcium. Upregulation of Dsg2 enhanced P-STAT3 level but not total STAT3 in transgenic compared with wild-type skin. Actin was used as a loading control. (B) Wild-type and transgenic keratinocytes were grown to confluency in low-calcium-containing CnT medium and then 1 mM calcium was added for 24 hours. Cells were fixed and stained with Flag and DG3.10 antibodies. Transgenic but not wild-type keratinocytes expressed the Dsg2-Flag protein. DG3.10 recognized the endogenous Dsg2 in the wild-type and as well as the Dsg2-Flag in the transgenic cells. (C) Wild-type and Inv-Dsg2 transgenic cells were trypsinized and put in suspension culture for up to 72 hours. Cells were collected at the time points indicated. Dsg2-Flag was detected by immunoblot analysis in Inv-Dsg2 cells within 24-72 hours in suspension culture. Immunoblotting analysis for actin expression showed equal loading. Similar results were observed with three independent wild-type and transgenic cell lines.
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