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. 2009 Jun 29;185(7):1243–1258. doi: 10.1083/jcb.200809044

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© 2009 Getsios et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 6. — Dsg1 promotes differentiation in the absence of robust PG binding. (A) To test which domains of Dsg1 would be sufficient to drive differentiation, we generated three Flag-tagged Dsg1 cDNA constructs: WT Dsg1 (Dsg1WT), a triple point mutant harboring three Ala substitutions (Dsg1AAA) within the predicted binding region for PG, or a truncation mutant lacking the ectodomain (ECTO) and transmembrane (TM) region (ΔN-Dsg1). CYTO, cytoplasmic domain. (B) The subcellular localization of Dsg1WT, Dsg1AAA, or ΔN-Dsg1 was determined in keratinocytes immunostained using a rabbit polyclonal antibody directed against Flag and a chicken polyclonal antibody against PG after exposing cells to high Ca²⁺ for 4 h to induce junction assembly. Both Dsg1WT and Dsg1AAA were efficiently recruited to areas of cell–cell contact; however, ΔN-Dsg1 was diffusely distributed throughout the cytoplasm. PG staining highlighted the intercellular borders; its localization at junctions was largely unaffected by any of the Dsg1 constructs. (C) Western blot analysis of keratinocytes transduced with these Dsg1 constructs and induced to differentiate for 2 d as submerged cultures. Although Dsg1WT and Dsg1AAA were sufficient to increase Dsc1/K10/loricrin, ΔN-Dsg1 did not affect these markers of differentiation compared with EGFP-transduced (Dsg1−) control cultures. FL, full length; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Bar, 20 µm.

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