FIG. 4.

Adding back NES-GSK-3β sensitizes neurons to apoptosis in GSK-3β-silenced cells. (A) GSK-3β shRNA-insensitive mutants (GSK-3βi) were prepared by introducing silent mutations within the shRNA-targeted sequence as shown. aa, amino acids. (B) Cerebellar granule neurons were transfected with GFP-GSK-3βi or GFP-NES-GSK-3βi, and subcellular localization was examined by confocal microscopy. Sections through nuclei are shown. (C) To determine the relative expression levels of nontargeted and NES-targeted GFP-GSK-3βi, cerebellar granule neurons were transfected as indicated, and lysates were immunoblotted for GFP. GFP-GSK-3βi and GFP-NES-GSK-3βi expression levels were equal. Nonspecific bands are indicated by an asterisk. (D) Cerebellar granule neurons (5 DIV) were cotransfected (+) with control shRNA or GSK-3β shRNA with (+) or without (−) targeted GFP-GSK-3βi or GFP-NES-GSK-3βi as shown. After 8 days of expression, neurons were deprived of trophic support for 20 h. Cells were stained with Hoechst 33342, and cells with pyknotic nuclei were scored as dead cells. Data are means plus standard errors of the means (error bars) from 5 to 10 replicates. Values that are significantly different from those of the corresponding control values are indicated (*, P < 0.05; ***, P < 0.001).