Figure 3
From: Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening
Dose-dependent inhibition of DLK-GFP localization and palmitoylation by ketoconazole. (A) Quantified DLK puncta per transfected cell from HEK293T cells transfected as in Fig. 2 and treated with the indicated concentrations of ketoconazole. Data (mean ± SEM) are plotted relative to DMSO vehicle control for 6 fields of view per condition from 8 determinations per condition. ***p < 0.001 compared to vehicle-treated control, ANOVA with post hoc Tukey test. F(6) = 14.6. Similar results were obtained in another experiment. (B) Left panels Images of HEK293T cells transfected with DLK-GFP cDNA and treated with 10 μM ketoconazole (‘Keto’) or vehicle, prior to fixation and immunostaining with anti-GFP and anti-GM130 antibodies. Scale bar: 10 μm. Right panel Quantified data from n = 22–24 cells per condition confirm that ketoconazole significantly reduces the fraction of DLK-GFP that colocalizes with GM130. *p < 0.05, t-test. (C) Palmitoyl-DLK levels (detected following Acyl-Biotin Exchange (ABE) assay to purify palmitoyl-proteins, top panel) from HEK293T cells transfected with DLK-GFP cDNA and subsequently treated with the indicated concentrations of ketoconazole. Middle and lower panels show total levels of DLK and tubulin, respectively, detected by western blotting of parent lysates. A negative control sample was generated by combining equal fractions of lysates from all conditions, which was then subjected to ABE in the absence of the key reagent hydroxylamine (HAM-). Molecular weight markers are indicated on each blot. (D) Quantified data from n = 3–7 determinations per condition from C confirm that ketoconazole significantly decreases palmitoyl:total DLK-GFP levels. **p < 0.01. ***p < 0.001 relative to vehicle treated cells, ANOVA with Bonferroni post hoc test, F(6) = 11.16. Some data from this panel are re-plotted in Fig. 4B.
