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. 2014 Nov 25;290(4):1927–1935. doi: 10.1074/jbc.M114.608703

FIGURE 3.

FIGURE 3.

SENP1 de-SUMOylates β-arrestin 2. A, SENP1 binds to β-arrestin 2. HEK293T cells were transfected with the indicated plasmids, and the transfected cell lysates were immunoprecipitated (IP) with anti-FLAG M2-agarose beads, followed by WB analysis with anti-ΗΑ or anti-FLAG antibodies. IB, immunoblot; WCL, whole-cell lysate. B, SENP1 deconjugates SUMOylated β-arrestin 2 in vivo. HEK293T cells were transfected with the indicated plasmids, and the transfected cell lysates were immunoprecipitated with anti-FLAG M2-agarose beads. The immunoprecipitates and the whole-cell lysates were analyzed by immunoblotting with anti-β-arrestin 2, anti-FLAG, or anti-RGS-his antibodies. MU, mutant. C, SUMOylated β-arrestin 2 is accumulated in Senp1−/− MEFs. MEF cell lysates were immunoprecipitated with anti-SUMO1 antibody or control IgG. The immunoprecipitates and cell lysates were analyzed by immunoblotting with anti-β-arrestin 2 or anti-SUMO1 antibodies. D, SENP1 enhances β-arrestin 2 inhibition of TRAF6 autoubiquitination. The indicated plasmids were transfected into HEK293T cells. The cell lysates were purified by Talon beads. The precipitates and cell lysates were immunoblotted using anti-FLAG, anti-HA, or anti-GFP antibodies. E, SENP1 enhances β-arrestin 2 inhibition of TRAF6-mediated NF-κB and AP-1 activation. The indicated plasmids were cotransfected into HEK293T cells with an NF-κB- or AP-1-dependent firefly luciferase reporter and a Renilla luciferase reporter. The relative luciferase activity was measured 36 h after transfection and normalized on the basis of Renilla luciferase activity. *, p < 0.05; **, p < 0.01. F, FLAG-TRAF6-transfected wild-type and Senp1−/− MEFs were treated with IL-1β or left untreated before harvest. The cell lysates were immunoprecipitated with anti-FLAG M2-agarose beads, followed by WB analysis with anti-ubiquitin or anti-TRAF6 antibodies. G, β-arrestin 2 WT- or β-arrestin 2 K295R-MCF-7 stable cells were transfected with HA-TRAF6 and si-SENP1 or si-NS oligonucleotides. The cells were treated with IL-1β before harvest, the cell lysates were immunoprecipitated with anti-HA-agarose beads, and the immunoprecipitates and the cell lysates were analyzed by immunoblotting with anti-ubiquitin, anti-HA, or anti-FLAG antibodies. H, wild-type and Senp1−/− MEFs were treated with IL-1β for the indicated time before harvest, followed by WB analysis with the indicated antibodies.