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

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex

doi: 10.1007/s00018-026-06146-9

Figure Lengend Snippet: ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with mouse anti-Flag mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments

Article Snippet: The following primary antibodies were obtained from commercial sources: mouse anti-Flag mAb (A00187 -100, GenScript), rabbit anti-Flag pAb (20543-1-AP, Proteintech) mouse anti-HA mAb (A01244-100, GenScript), rabbit anti-HA pAb (51064-2-AP, Proteintech), mouse anti-V5 mAb (A01724-100, GenScript), rabbit anti-V5 pAb (14440-1-AP, Proteintech), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech), rabbit anti-ENO1 pAb (11204-1-AP, Proteintech), rabbit anti-LaminA/C pAb (10298-1-AP, Proteintech), rabbit anti-KPNA4 pAb (12463-1-AP, Proteintech), rabbit anti-PB2 pAb (GTX125926, GeneTex), rabbit anti-PB1 pAb (GTX125923, GeneTex), rabbit anti-PA pAb (GTX118991, GeneTex), rabbit anti-NP pAb (GTX125989, GeneTex).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Virus, Purification, Staining