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. 2019 Oct 4;294(45):16527–16534. doi: 10.1074/jbc.AC119.010671

Figure 2.

Figure 2.

Ubiquitin RING E3 ligase RNF186 controls Sestrin-2 ubiquitination and degradation. A, screening of an siRNA library targeting ubiquitin E3 ligases for effect on Sestrin-2-GFP signal. Fluorescence was measured by Cytation 5 automated microcopy. Sestrin-2-GFP signal was reported as median absolute deviation Z-score. Top hits are ranked and displayed. B, representative Sestrin-2-GFP images from scramble siRNA and candidate E3 ligase siRNA from screen in A. Scale bar, 100 μm. C, immunoblot analysis of candidate E3 ligases identified from the screen in A. D, RNF186 was expressed dose-wise in Beas-2b prior to Sestrin-2 immunoblotting. E, immunoblot analysis of Sestrin-2 following expression of RNF186 H60W mutant. F, CHX chase of Sestrin-2 protein following siRNA knockdown of RNF186 in Beas-2b cells. Shown are data and means ± S.D. (error bars) of three independent experiments. G, CHX chase of Sestrin-2 protein following expression of RNF186 in Beas-2b cells. Shown are data and means ± S.D. of three independent experiments. H, immunoblot analysis of Sestrin-2 ubiquitination following in vivo ubiquitination assay and pulldown of Sestrin-2 protein. *, p < 0.05, by F-test comparisons of nonlinear regression compared with controls (F and G). WCL, whole-cell lysate.