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. 2017 Jun 16;24(9):1530–1539. doi: 10.1038/cdd.2017.29

Figure 5.

Figure 5

NR4A1 expressed in response to TPA stimulation undergoes SUMOylation-mediated degradation. (a) HEK293T cells were treated with DMSO or the proteasome inhibitor MG132 (5 μM for 5 h) along with TPA (100 ng/ml) for the indicated time. Cells were collected for immunoblot (IB) analysis. (b) HEK293T cells were treated with TPA (100 ng/ml) and MG132 (5 μM) as indicated for 5 h. Cells were collected for immunoprecipitation (IP) with non-specific (ns) or anti-NR4A1 antibody followed by IB analysis. (c) HEK293T cells were transfected with control vector, SUMO2, or SUMO3 plasmids as indicated. Cells were treated with TPA for the indicated time and collected for IB analysis. (d) PIAS3 WT but not the PIAS3▴Ring mutant accelerated the degradation of the induced NR4A1 in HEK293T cells. (e) IB analysis of cell lysate from HEK293T cells transfected with control vector, SENP1 WT, or the SENP1 CS mutant and treated with TPA (100 ng/ml) for the indicated time. (f) RNF4 depletion using two independent shRNAs (#1 and #2) increased and extended TPA-induced NR4A1 expression in Jurkat cells. shRNF4, small hairpin RNA-targeting RNF4. NS, non-specific. Actin was included as a loading control in (a) and in (c–f)