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. 2016 Feb 15;27(4):617–626. doi: 10.1091/mbc.E15-10-0703

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© 2016 Xiao et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — SPRY4-IT1 silencing enhances TJ mRNA decay and represses their translation. (A) Levels of claudin-1, occludin, claudin-3, and JAM-A mRNAs 48 h after transfection with siSPRY4-IT1 or C-siRNA. Values are means ± SEM from three separate experiments. *p < 0.05 compared with C-siRNA. (B) Stability of the TJ mRNAs in cells described in A. mRNA levels were examined at different times after administration with actinomycin D. (C) Newly synthesized TJ proteins in SPRY4-IT1–silenced cells. After cells were exposed to AHA, cell lysates were incubated with the reaction buffer containing biotin/alkyne reagent; the biotin-alkyne/azide–modified protein complex was pulled down by paramagnetic streptavidin-conjugated Dynabeads. (D) Distribution of claudin-1, occludin, claudin-3, and JAM-A mRNAs in each gradient fraction prepared from polysomal profile after SPRY4-IT1 silencing. Nuclei were pelleted, and the resulting supernatants were fractionated through a 10–50% linear sucrose gradient. Total RNA was isolated from different fractions, and the levels of TJ and Gapdh mRNAs were measured and plotted as a percentage of each of total TJ mRNAs and Gapdh mRNA levels in the samples. Three experiments were performed and showed similar results.