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. 2008 Oct 24;283(43):29424–29432. doi: 10.1074/jbc.M802475200

FIGURE 3.

FIGURE 3.

SCFβ-TRCP regulates Plk2-dependent SPAR abundance, turnover, and promotes its ubiquitination. A and B, depletion of β-TRCP by RNAi protects GFP-SPAR from degradation in individual HEK293T cells. The indicated plasmids were transfected with pCMV-RFP as a co-transfection marker into HEK293T cells. After 96 h, cells were fixed and imaged for GFP-SPAR and RFP expression (A) and quantified as the percentage of RFP expressing cells that also expressed GFP-SPAR (B). Values represent the mean ± S.E. from three independent experiments, derived from analysis of 400 cells per experiment per condition (n = 1200 cells per condition). **, p < 0.01; NS, not significant; one-way analysis of variance, compared with control condition (pRSP+Plk2D/A). C and D, depletion of β-TRCP by RNAi stabilizes SPAR abundance and turnover in the presence of Plk2 activity. HEK293T cells were transfected with vectors expressing myc-SPAR (0.5 μg), WT, or catalytically inactive HA-Plk2 (D/A) (1 μg), and the indicated shRNA vector carrying a puromycin resistance selection marker (2.5 μg). 36 h post-transfection, cells were incubated with media containing 1 μg/ml puromycin to enrich for shRNA expression. Extracts were subsequently examined by immunoblotting 96 h post-transfection as indicated. Endogenous Cdc25A was probed to control for successful knockdown of β-TRCP. In panel D, HEK293T cells were transfected in an identical fashion to panel C, except that 24 h post-transfection the transfected cells were split among 5 wells in puromycin-containing media. After 48 h of puromycin selection, cells were treated with 25 μg/ml cycloheximide (CHX) and harvested at the indicated times before immunoblotting. E, expression of β-TRCP promotes Plk2-mediated SPAR ubiquitination. HEK293T cells were transfected with myc-SPAR (1 μg), His-Ub (1 μg), HA-Plk2 (1.5 μg, wild type or D201A), and GST or GST-β-TRCP (4.5 μg). Twenty hours post-transfection cells were treated with 25 μm MG-132 for 5 h and lysed in buffer containing 10 mm N-ethylmaleimide. Myc-SPAR was purified with c-Myc 9E10-agarose, resolved on 6% Tris-glycine SDS-PAGE gel, and immunoblotted (IB) with anti-Myc antibodies. IP, immunoprecipitates.