Figure 5. Inhibitors targeting MAP4K4, TNIK, and MINK1 block DLK/JNK pathway activation in axons and induction of DLK and p-JNK in the soma after NGF withdrawal. A, Schematic illustration of experimental setup for isolation of distal axons (lower compartment, orange) from DRG cell bodies/proximal axons (inner compartment, blue) of DRG explants. Western blot analysis of DRG cell bodies/proximal axons (left, blue) and axons (orange, right), demonstrates localization of MAP4K4, MINK1, and TNIK in isolated axons as well as cell bodies. B–G, Immunoblot analysis of isolated DRG axons and cell bodies after NGF-withdrawal at 1, 3, and 6 h in the presence of DMSO or GNE-495. B–D, The presence of MAP4Ki GNE-495 reduced phosphorylation of c-Jun in explants (blue) (C) and JNK in explants and axons (red) (D) after NGF deprivation compared with DMSO control. Immunoblot intensities were normalized to TUJ1 and +NGF control (0 h of NGF withdrawal). p-c-Jun: n = 4–5/condition, p-JNK: n = 8–9/condition. E, Immunoblot analysis of DLK protein levels and p-c-Jun in isolated DRG axons and cell bodies at 1, 3, and 6 h in the presence of DMSO or GNE-495 (top). Illustration at the bottom shows how the activated fraction of DLK was calculated based on the Western blot banding pattern: molecular-weight-shifted DLKshift (red) divided by total DLKtotal (blue) normalized to actin immunoblot band intensity. The dashed line highlights the magnified region of full blot. F, Total DLK protein levels increase in cell bodies/proximal axons over time, but decrease in axons. GNE-495 block DLK induction in explants (n = 7–8/condition). G, Active fraction of DLK protein (DLKactive, see example illustration in E) is increased significantly in axons and cell bodies/proximal axons over time. GNE-495 treatment prevents DLK activation in both axons and explants (n = 7–8/condition). H, Immunofluorescence images demonstrating that both p-JNK (green) and DLK (red) protein is increased in the DRG cell bodies (DAPI: blue, TUJ-1: white) 3 h after NGF withdrawal. MAP4K inhibitors block the somal intensity of DLK and p-JNK. I, Quantification of somal p-JNK intensity relative to NGF p-JNK intensity in experiment H. J, Quantification of somal DLK intensity relative to NGF DLK intensity in experiment H (n = 40 cells/condition, obtained from 4 independent wells). One-way ANOVA followed by Bonferroni post hoc test was used for statistical comparison. Data are represented as mean ± SEM. Statistical significance level between treatment for all time points are displayed above graphs in C, D, F, and G; statistical significance level of individual time points are presented in the graphs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.