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. 2015 Jun 29;212(7):1021–1041. doi: 10.1084/jem.20150354

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© 2015 Watson 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.rupress.org/terms). 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 8. — CD99 cytoplasmic tail mediates TEM through a positively charged juxtamembrane region. (a) Diagram of CD99 cytoplasmic tail and CD99-GFP constructs. (b) Endogenous CD99 in HUVECs was knocked down using CD99 shRNA. CD99-GFP constructs were then reexpressed. Samples treated in parallel were lysed and used for immunoblot analysis of CD99 to assess degree of reexpression and knockdown. (c) Quantitative TEM assays were then performed on knockdown samples. (d) CD99-GFP constructs were overexpressed in iHUVECs. CD99 was then immunoprecipitated from the surface of cells (ensuring only fully processed CD99 was being analyzed). Immunoblot analysis for ezrin, sAC, and CD99 was then performed. (e and f) Quantification of results above. Amount of ezrin (e) and sAC (f) in each sample was normalized to the amount of CD99 immunoprecipitated (to account for any variability in pull-down efficiency between samples). Values were then normalized to wild-type CD99-GFP condition. Images are representative three (b and d) independent experiments. Numerical values are the mean of three (c, e, and f) independent experiments. Error bars represent SD (e and f) or SEM (b; ***, P < 0.001; ****, P < 0.0001; ANOVA).