Figure 4
From: Identification of Novel Inhibitors of DLK Palmitoylation and Signaling by High Content Screening
Ketoconazole inhibits palmitoylation of DLK and PSD-95, but not GAP43. (A) HEK293T cells were transfected with DLK-GFP cDNA and incubated with 20 µM 2BP, or with 2.5 µM or 5 µM ketoconazole 2 h post-transfection for 16–18 h. Upper western blot shows DLK total expression and palmitoyl-DLK levels (from ABE, ‘HAM+’) for each condition. 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-). Lower western blot shows tubulin levels, an indication of total protein expression. Molecular weight markers are indicated on each blot. Differing band widths in this panel are likely due to different protein concentrations and/or ionic strength of total lysates versus ABE fractions. (B) Histogram of pooled data (mean ± SEM) for 4 determinations per condition from A. Ketoconazole and 2BP both significantly reduce DLK palmitoylation. Some data from this panel are re-plotted as part of Fig. 3D. (C) As A, except that cells were transfected with GAP43-Myc cDNA ABE fractions were blotted with anti-Myc antibody and cell lysates were blotted to detect total expression of GAP43-myc (upper panel) and GAPDH (lower panel). (D) Histogram of pooled data (mean ± SEM) for 7 determinations per condition from C. Ketoconazole does not reduce GAP43 palmitoylation, but 2BP does. (E) As C, except that cells were transfected with PSD-95 cDNA and total lysates and ABE fractions were blotted with anti-PSD-95 antibody. (F) Histogram of pooled data (mean ± SEM) for 4 determinations per condition from E. 5 μM ketoconazole and 2BP both reduce PSD-95 palmitoylation. One-way ANOVA, Kruskal-Wallis post-hoc analysis; (A) ANOVA p = 0.0009, h = 13.92, (C) ANOVA not significant, (E) ANOVA p = 0.0158, h = 8.290.
