Fig 1: Targeted inhibition of APT2 inhibits ultraviolet B (UVB)-induced melanomagenesis in vivo. a Illustration of the dynamic melanocortin-1 receptor (MC1R) palmitoylation/depalmitoylation cycle. b cAMP levels in human primary melanocytes (HPMs) after treatment with increasing concentrations of the indicated inhibitors. HPMs in which endogenous MC1R is stably depleted using shMC1R were infected with Flag-MC1RR151C and then treated with 1 μM α-melanocyte-stimulating hormone (α-MSH) and indicated inhibitors for 3.5 h. The resulting cells were harvested for a cAMP immunoassay. The data were compiled from five independent experiments. c MC1R-depleted HPMs were infected with the indicated Flag-MC1R-encoding retroviruses and then pretreated with 1 μM α-MSH and 100 nM ML349 for 30 min followed by 100 J/m2 UVB irradiation. The resulted cells were harvested for immunoprecipitation, acyl-biotin exchange, and immunoblotting analysis with the specific antibodies as indicated 3 h after UVB exposure. d cAMP levels in MC1R-depleted HPMs expressing the indicated Flag-MC1R encoding retroviral constructs and pretreated with 1 μM α-MSH and 100 nM inhibitors for 30 min followed by 100 J/m2 UVB irradiation. The resulted cells were harvested for cAMP immunoassay 3 h after UVB exposure. Data were compiled from five independent experiments. e–g Growth curves (e), dissected tumors (f), and tumor weight (g) for the xenograft experiments. MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAFV600E melanocytes were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were preincubated with 1 μM α-MSH and 100 nM inhibitors for 30 min before being irradiated with 20 J/m2 UVB. After 24 h, 3 × 106 cells were inoculated subcutaneously into each flank of nude mice. Visible tumors were measured at the indicated days. h Melanoma-free survival of the indicated mice. In the UVB radiation period, 5 mg/kg ML349 was injected intraperitoneally into mice prior to the treatment with UV. Ninety days after the final UVR, melanoma was diagnosed in 23% (3/13) or 64% (7/11) of mice with or without ML349 treatment, respectively (p = 0.0366). **p < 0.01, ***p < 0.001, unpaired Student’s t test. Error bars represent ± s.d.
Fig 2: Depalmitoylating enzyme activity is unchanged following cLTP. (A) Representative images of the fluorescent depalmitoylation probe-5 (DPP-5; 2 μM) 1 h after cLTP treatment in 14 DIV-cultured hippocampal neurons transfected at 11 DIV with mCherry. Left: mCherry cell fill. Middle: Background fluorescence within mCherry mask prior to addition of DPP-5 to the bath. Right: DPP-5 fluorescence within the mCherry mask 50 mins post addition of DPP-5 to the bath (1 h post cLTP). Scale bars: 100 μm. (B) As A, but 24 h post cLTP treatment. (C) Graph of time-course of DPP-5 fluorescence increase (ΔF) following cLTP and subsequent bath addition of DPP-5. No significant difference was observed between mock- and cLTP-treated neurons at any time point. (D) Graph of DPP-5 fluorescence increase (ΔF) 1 h post cLTP and 50 min post addition of DPP-5 to the bath. No significant difference was observed between mock and cLTP-treated neurons at any time-point. Mock, n=16 neurons; cLTP, n=17 neurons from two independent cultures. (E,F) As C,D but 24 h post cLTP and 50 min post addition of DPP-5 to the bath. Mock, n=17 neurons; cLTP, n=14 neurons from three independent cultures. AU, arbitrary units. (G) Acyl-Rac assay showing palmitoylated ABHD17 in cultured hippocampal neurons (14 DIV) following cLTP. Palmitoylated ABHD17 values in the graph were derived from ABHD17 ‘palm’ normalized to ABHD17 ‘input’ and the β-actin loading control. n=3 independent hippocampal cultures per condition. (H) Phospho-protein purification assay showing phosphorylated ABHD17 following cLTP. Phosphorylated ABHD17 values in the graph were derived from ABHD17 ‘phospho’ normalized to ABHD17 ‘input’ and the β-actin loading control. n=3 independent hippocampal cultures per condition. (I) Acyl-Rac assay showing palmitoylated APT2 in cultured hippocampal neurons (14 DIV) following cLTP. Palmitoylated APT2 values in the graph were derived from APT2 ‘palm’ normalized to APT2 ‘input’ and the β-actin loading control. n=3 independent hippocampal cultures per condition. (J) Phospho-protein purification assay showing phosphorylated APT2 following cLTP. Phosphorylated APT2 values in the graph were derived from APT2 ‘phospho’ normalized to APT2 ‘input’ and the β-actin loading control. n=3 independent hippocampal cultures per condition. n=3 hippocampal cultures per experiment. For all graphs, results are mean±s.e.m. with individual data points shown. ns, not significant (unpaired two-tailed Student's t-test for D,F; one-way ANOVA with Tukey's post hoc test for G–J).
Supplier Page from Abcam for Anti-APT-2 antibody