Article Figures & Data
Figures
- Figure 1.
Phosphorylation of eIF2α upon AP20187-induced FKBP-PKR dimerization. A, Schematic diagrams showing the fusion of PKR kinase domain with AP20187-binding domain of FKBP. Application of AP20187 induces the dimerization of the PKR fusion proteins and activates PKR, triggering eIF2α phosphorylation and subsequent de novo protein synthesis inhibition. B, Representative blots showing AP20187-induced phosphorylation of eIF2α on Ser51 in HEK293T cells expressing HA-FKBP-PKR. Western blotting was performed using specific antibodies as indicated. The blots were also probed with an anti-tubulin antibody for loading controls (Ctr.). C, Time course of eIF2α phosphorylation induced by AP20817. Multiple blots were quantified (N = 3), and eIF2α-P signals at various time points were normalized to that at “0” h.
- Figure 2.
Inducible inhibition of protein synthesis by the FKBP-PKR dimerization system. A–D, PKR-mediated inhibition of exogenous cytosolic protein (destabilized EGFP) level and endogenous membrane protein (TfR) level. HEK293T cells or rat cortical neurons were transfected with destabilized EGFP alone (A, C) or in combination with FKBP-PKR (B, D) and treated with AP20187 at various time intervals. Relative levels of proteins were quantified by Western blots. N = 3 independent experiments. Ctr., Control.
- Figure 3.
Inhibition of BDNF-mediated spine growth by the FKBP-PKR system. A, Sample images of cultured hippocampal neuronal dendrites under various conditions. Rat hippocampal neurons (18–21 d in vitro), transfected with GFP or FKBP-PKR, were fixed after indicated treatments. Scale bar, 10 μm. B, C, Role of protein synthesis in BDNF regulation of dendritic spines and filopodia. Activation of PKR by AP20187 blocks BDNF-induced changes in spines (B) and filopodia (C). At least three independent cultures were examined. Error bars are SEM, and numbers in the columns are the numbers of neurons examined. *p < 0.01, ANOVA followed by post hoc test.
- Figure 4.
Requirement of protein synthesis in CA1 but not CA3 neurons in hippocampal L-LTP. GFP or FKBP-PKR-GFP Sindbis virus was injected into either CA1 or CA3 region of hippocampus of 8-week-old C57BL/6 mice. Approximately 36–48 h after in vivo injection, the animals were killed and hippocampal slices were prepared. A, Localization of FKBP-PKR, as indicated by GFP fluorescence, in the CA1 area. The arrowhead indicates the viral injection site. B, Normal E-LTP in FKBP-PKR injected slices treated with vehicle or AP20187. Sample traces before (“1”) and 3 h (60 min for B) after (“2”) TBS stimulation in this and other figures are shown on top of each figure. C, Inhibition of L-LTP by AP20187 in slices expressing FKBP-PKR in CA1. L-LTP was induced by 12-TBS. The difference between vehicle- (155 ± 8%, SEM) and AP20187- (104 ± 3%) treated slices at “2” is highly significant (p < 0.0002, t test). D, Normal L-LTP in AP20187-treated slices expressing FKBP-PKR in CA3.
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Figures
