Fig 1: ZDHHC activity-dependent post-translational changes in vivo. Nine-week-old male mice were subjected to contextual fear conditioning (cFC) and hippocampal lysates from conditioned and unconditioned mice were collected 1 h later. (A,C,E,G) Lysates were run through the phospho-protein purification assay and western blots probed with antibodies for endogenous ZDHHC5, ZDHHC8, ZDHHC9 and ZDHHC2. Phosphorylated ZDHHC values in the graphs were derived from ZDHHC ‘phospho’ normalized to ZDHHC ‘input’ and the β-actin loading control. Input fractions were quantified to assess overall ZDHHC protein levels in control and cFC lysates. (B,D,F,H) Acyl-Rac assay showing palmitoylated ZDHHC5, ZDHHC8, ZDHHC9 and ZDHHC2 in control or cFC-treated mice. Palmitoylated ZDHHC values in the graphs were derived from ZDHHC ‘palm’ normalized to ZDHHC ‘input’ and the β-actin loading control. β-actin loading controls are duplicated in some panels as individual blots were cut in half and probed for two ZDHHCs per blot. Cut blots were probed for either ZDHHC2 (bottom half)/ZDHHC5 (top half) or ZDHHC9 (bottom half)/ZDHHC8 (top half). Con 1 and Con 2 represent two biological replicates from the control group. FC 1 and FC 2 represent two biological replicates from the cFC-treated group. ns, not significant; *P<0.05 (unpaired two-tailed Student's t-test). Results are mean±s.e.m. with individual data points shown. n=5 hippocampi per experiment.
Fig 2: The palmitoylation of ZDHHC9 and its substrates are reduced following cLTP. (A) Western blot analysis of ZDHHC9 protein levels in primary hippocampal neuron cultures 40 min, 2 h and 24 h following cLTP. n=5 independent hippocampal cultures per condition. (B) Acyl-Rac assay showing palmitoylated ZDHHC9 and overall ZDHHC9 protein (input) levels following cLTP. Palmitoylated ZDHHC9 values in the graph were derived from ZDHHC9 ‘palm’ normalized to ZDHHC9 ‘input’ and the ß-actin loading control. n=3 independent hippocampal cultures per condition. (C) Lysates were run through the phospho-protein purification assay and western blots probed with the anti-ZDHHC9 antibody showing phosphorylated ZDHHC9 and overall ZDHHC9 protein (input) levels following cLTP. Phosphorylated ZDHHC9 values in the graph were derived from ZDHHC9 ‘phospho’ normalized to ZDHHC9 ‘input’ and the ß-actin loading control. n=3 independent hippocampal cultures per condition. (D) Cells were nucleofected at the time of plating with ZDHHC9 shRNA to knock down ZDHHC9, and with HA–ZDHHC9-WT or HA–ZDHHC9-DHHS9. At 13–15 DIV, the cultures were stimulated using cLTP treatment and HA–ZDHHC9 palmitoylation was determined using the Acyl-Rac assay. n=3 independent hippocampal cultures per condition. (E,F) At 14 DIV, the cultures were stimulated using cLTP treatment and N-RAS (E) or TC10 (F) palmitoylation was determined using the Acyl-Rac assay. n=3 independent hippocampal cultures per condition. For all graphs, results are mean±s.e.m. with individual data points shown. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. For A–D, P-values were determined versus the first condition in the bar chart (one-way ANOVA with Tukey's post hoc test). For E,F, P-values were determined using unpaired two-tailed Student's t-test. (G) Schematic showing activity-induced decrease in ZDHHC9 and substrate palmitoylation.
Supplier Page from MilliporeSigma for Anti-ZDHHC9 antibody produced in rabbit