Journal: The Journal of Biological Chemistry
Article Title: The protein phosphatase 2A-B56α complex regulates N-Myc degradation in neuroblastoma
doi: 10.1016/j.jbc.2026.111298
Figure Lengend Snippet: PP2A activity is dependent on the N-Myc phosphorylation sites S62 and T58. A , Kelly cells were transduced with pLenti6.3 expressing V5-tagged WT N-Myc and mutant S62D or T58A N-Myc. Cells were treated with 25 μg/ml cycloheximide in a chase experiment to assess protein stability over 60 min by immunoblot of whole cell lysate for V5-tagged N-Myc. B , quantification of V5-tagged N-Myc plotted after normalization to Vinculin and the untreated control. Data plotted as mean ± S.D. are from four independent experiments ( n = 4), and half-lives were determined from exponential decay curve fitting. C , Kelly cells expressing the WT, S62D, and T58A N-Myc are treated with DMSO or 20 μM DT-061 for 3 h and immunoblotted for V5-tagged N-Myc, representative images are presented. D , quantification of V5-tagged N-Myc after normalization to Vinculin and DMSO controls from three independent experiments ( n = 3). Statistical significance was determined by two-way ANOVA; ∗ p < 0.05; ns, not significant. E , representative images of clonogenic assays of Kelly WT and S62D N-Myc plated at low density and treated with DT-061 for 14 days. F , colony formation assays from panel E were quantified by absorbance of dissolved crystal violet stained colonies, and plotted data represent the mean ± S.D. of three independent biological replicates (two technical replicates for each biological replicate).
Article Snippet: Membranes were blocked in 5% non-fat milk/TBST for 1 hour and incubated overnight at 4 °C with primary antibodies: c-Myc (ABclonal, A19032, 1:1000), phospho S62 c-Myc (Abcam, ab185656, 1:1000), N-Myc (Abcam, ab16898, 1:1000), Vinculin (Santa Cruz Biotech, sc-73614, 1:5,000), GAPDH (Santa Cruz Biotech, sc-32233, 1:2000), V5-tag (Cell Signaling Technology, 13,202, 1:1000), AP2β (Cell Signaling Technology, 2509, 1:1000), GATA3 (Cell Signaling Technology, 5852, 1:1,000), PHOX2B (Santa Cruz Biotech, sc-376997, 1:1,000), PARP (Cell Signaling Technology, 9542L, 1:1000), Cleaved caspase-3 (Cell Signaling Technology, 9664L, 1:1,000), and PPP2R2A (Santa Cruz Biotech, sc-81606, 1:1000).
Techniques: Activity Assay, Phospho-proteomics, Transduction, Expressing, Mutagenesis, Western Blot, Control, Staining
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