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. 2012 Apr 18;287(27):22882–22888. doi: 10.1074/jbc.M111.306373

FIGURE 4.

FIGURE 4.

Overexpression of GSK3β enhances ZAP activity, whereas down-regulation of GSK3β reduces ZAP activity. A, 293TRex cells were cotransfected with a plasmid expressing wild-type ZAP or ZAP(S1–8A) and the ZAP-responsive reporter pMMLV-luc. At 6 h post-transfection, cells were mock-treated or treated with 1 μg/ml tetracycline to induce ZAP and ZAP(S1–8A) expression. Cells were lysed, and luciferase activities were measured at 48 h post-transfection. Relative -fold inhibition was calculated as the firefly luciferase activity with ZAP divided by that with ZAP(S1–8A). Data are presented as means ± S.D. of three independent experiments (upper panel). The expression of ZAP and ZAP(S1–8A) was confirmed by Western blotting (lower panel). B, 293TRex-ZAP cells were cotransfected with the indicated amounts of the GSK3β-expressing plasmid, the ZAP-responsive reporter pMMLV-luc, and the internal control pRL-TK. At 6 h post-transfection, cells were mock-treated or treated with 1 μg/ml tetracycline to induce ZAP expression. Cells were lysed, and luciferase activities were measured at 48 h post-transfection. The firefly luciferase activity expressed from pMMLV-luc was normalized by the Renilla luciferase activity expressed from pRL-TK. -Fold inhibition was calculated as the normalized luciferase activity in the mock-treated cells divided by that in the tetracycline-treated cells. Relative -fold inhibition was calculated as the -fold inhibition with GSK3β divided by that without GSK3β (upper panel). Data are presented as means ± S.D. of three independent experiments. The expression of ZAP and GSK3β was confirmed by Western blotting (lower panel). C, plasmids expressing the indicated FLAG-tagged proteins and shRNAs were cotransfected into HEK293 cells. Protein expression levels were measured by Western blotting at 48 h post-transfection. D, 293TRex-ZAP cells (left panel) and 293TRex-ZAP(S1–8A) (right panel) were transfected with the plasmids indicated together with pMMLV-luc and pRL-TK. Relative -fold inhibition (F.I.) was calculated as described for B. Data are presented as means ± S.D. of three independent experiments. GSK3βM is the GSK3β-expressing plasmid that is not targeted by Gi-5. hGSK3β, human GSK3β; EV, pSUPER.retro empty vector; Ctrl, control. *, p < 0.05.