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. 2009 May 5;284(26):17411–17419. doi: 10.1074/jbc.M109.016170

FIGURE 3.

FIGURE 3.

GSK-3β controls GCM1 ubiquitination and degradation. A, GCM1 ubiquitination is regulated by GSK-3β. 293T cells were transfected with 1 μg of pGCM1-FLAG, pHA-Ub, and the indicated GSK-3β expression plasmid. At 24 h post-transfection, cells were treated with 40 μm MG132 for an additional 4 h and then subject to ubiquitination analysis by immunoprecipitation (IP) with FLAG mAb and immunoblotting (IB) with HA mAb (left). In a separate experiment, 293T cells were transfected with 1 μg of pGCM1-FLAG and pHA-Ub plus 10 nm GL2 or GSK-3β siRNA and incubated as above for ubiquitination analysis. B, the half-life of GCM1 is prolonged by inhibition of GSK-3β. 293T cells were transfected with 0.35 μg of pGCM1-FLAG. At 24 h post-transfection, cells were pretreated with or without 50 mm LiCl for 2 h before incubation with 75 μm cycloheximide for the indicated period of time. In a separate experiment, 293T cells were transfected with 0.35 μg of pGCM1-FLAG plus 10 nm GL2 or GSK-3β siRNA. At 24 h post-transfection, cells were incubated with 75 μm cycloheximide for the indicated period of time. Cells were then harvested for immunoblotting with FLAG or α-tubulin antibody.