Figure 8.

NES-JBD selectively blocks cytosolic JNK in cortical neurons. The compartmental specificity of GFP-NES-JBD was tested. (A) 1-d-old cortical neurons were transfected with the JNK inhibitor pEGFP-NES-JBD and pEGFP–NES–c-Jun as shown. Samples were immunoblotted with phospho–S63–c-Jun (P–c-Jun) or GFP antibodies. (B) Quantitative data from A, showing means ± SEM (n = 3). (C) Cortical neurons were transfected with pEBG-JNK1, pEGFP-NES-JBD, and pECFP-SCG10. GST-JNK1 was isolated and kinase activity toward recombinant GST–c-Jun measured. (D) To test whether GFP-NES-JBD interfered with nuclear events, cerebellar granule neurons were transfected with a GAL4-luciferase reporter, GAL4–c-Jun (5–105), Renilla luciferase internal control, and pEGFP-NES-JBD or empty vector (pCMV) as indicated. Trophic withdrawal was for 4 h. Firefly luciferase normalized to Renilla luciferase activity was expressed as arbitrary units. Means ± SEM (n = 3) are shown. GFP-NES-JBD did not inhibit nuclear reporter activity.