FIG. 5.

GSK-3β is required for Hdm2-mediated cytoplasmic shuttling of p53 in ER-stressed cells. (A) Subcellular localization of GFP-p53 in GSK-3β^−/− MEFs. Cells were transfected with 0.05 μg of GFP-p53 WT cDNA, 0.05 μg of Hdm2 cDNA, and 0.1 μg of either GSK-3β WT or GSK-3β KD cDNA. After 24 h, cells were treated with either 10 μg of TM/ml or 1 μM TG for 4 h and then examined for GFP-p53 localization by fluorescence microscopy. The cell nuclei were visualized by DAPI staining. Quantification of GFP-p53 localization was performed as described in Fig. 2A and Materials and Methods. (B) Subcellular localization of GFP-p53 in 2KO MEFs. Cells were transiently transfected with 0.25 μg of GFP-p53 cDNA in the absence or presence of 0.25 μg of Hdm2 of WT cDNA and 0.5 μg of either GSK-3β WT cDNA or GSK-3β KD cDNA. After 24 h, cells were left untreated or treated with either 10 μg of TM/ml or 1 μM TG for 4 h, followed by examination with fluorescence microscopy. Quantification of the subcellular localization of GFP-p53 was performed as described above.