Fig. 3.

The JNK-specific peptide inhibitor d-JNKI-1, which blocks the interaction of JNK with its substrates, decreased NGF-induced neurite outgrowth and NGF-induced interaction of JNK with cytoskeletal proteins and RNA transport granule proteins. A, PC12 cells were pretreated for 1 h with different concentrations of d-JNKI-1 or the control peptide d-TAT before NGF stimulation. Assessment of neurite elongation after 24 h revealed that d-JNKI-1 dose-dependently decreased neurite outgrowth by ∼60% at a concentration of 10 μm. The control peptide d-TAT had no effect on neurite outgrowth. **p < 0.01, ***p < 0.001 versus NGF + d-TAT (n = 3 each). Scale bar: 20 μm. B, PC12 cells were grown in light, medium, or heavy media and were stimulated with NGF for 3 h. Medium-labeled cells were pretreated for 1 h with d-JNKI-1. The heavy-labeled cells were pretreated with the control peptide d-TAT. After JNK immunoprecipitation (IP), samples were combined and analyzed via LC-MS/MS. C, 701 proteins were quantified with at least three ratio counts. PANTHER analysis showed an enrichment for cytoskeletal proteins (n = 65) and nucleic acid binding proteins (n = 94), which specifically co-immunoprecipitated with JNK after NGF treatment. A log₂ fold change of 0.7 was applied as a cutoff. The three most significant PANTHER terms per cluster are shown. Highlighted proteins are discussed in the “Results” section.