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. 2018 Nov 2;293(52):20099–20111. doi: 10.1074/jbc.RA118.005802

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© 2018 Zhu et al.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 2. — RNF31 is a positive regulator for the stability of human FOXP3 protein. A, RNF31 was knocked down in primary human Treg cells, and the endogenous FOXP3 level was visualized by IB. B, RNF31 was knocked down with shRNA-bearing lentiviruses in primary Treg cells, the mRNA level of CD25, FOXP3, were detected (n = 4); *, p < 0.05; **, p < 0.01; ***, p < 0.01. C, RNF31 was knocked down in the stable cell line, FLAG-FOXP3 Jurkat T cells, and the level of FLAG-FOXP3 was then detected by Western blotting. D, RNF31 was knocked down in FLAG-FOXP3 Jurkat T cells, and the mRNA level of FOXP3 was investigated (n = 2). ns, not significant. E and F, immunoblot analysis of FLAG-FOXP3 in HEK-293T cell lysate co-transfected with FLAG-FOXP3 and various doses of Myc-RNF31. G and H, immunoblot analysis for identifying the half-life of FLAG-FOXP3 in HEK-293T cells transfected with FLAG-FOXP3 and Myc-RNF31. I, immunoblot analysis for investigating the role of HA-RNF31 in Myc-STUB1–mediated protein degradation of FLAG-FOXP3 in HEK-293T cells transfected with FLAG-FOXP3, HA-RNF31, and Myc-STUB1.