Fig. 4: ERK1/2 phosphorylates YTHDF2 at serine 39 and threonine 381 to stabilize YTHDF2.
a In vitro kinase assay of GST-YTHDF2. Recombinant active ERK1 and wild type (WT), S39A, T381A, or S39A/T381A YTHDF2 were incubated in the presence of [γ-32P] ATP. Proteins were separated by SDS-PAGE, transferred to PVDF membrane and autoradiography performed. MBP was included as positive control and co-incubated with SCH772984 as negative control. Coomassie blue stain was shown as loading control. Representative image of three independent experiments. b Stability of wild type or S39A/T381A mutated Flag-YTHDF2 was measured by CHX (50 μg/mL) chase assay in LN229/EGFRvIII cells cultured with Dox (10 μg/ml) (left panel) and analysis of YTHDF2 protein lifetime (right panel). Data are mean ± S.D. of three independent experiments. (unpaired two-sided t test). c In vitro invasion assay for LN229 cells transfected with wild type or S39A/T381A mutated Flag-YTHDF2. Representative invasion images of the cells (left panel) and quantitation of relative cell invasion (right panel). Data are mean ± S.E.M., n = 4 biologically independent experiments (one-way ANOVA Tukey’s post hoc test). Scale bar = 50 μm. d, e Cell viability of wild type or S39A/T381A mutated YTHDF2 expressing GSC11 (e) or GSC7-2 (f) cells with YTHDF2 depletion was measured by CCK-8. Data are mean ± S.E.M., n = 6 biologically independent experiments (one-way ANOVA Tukey’s post hoc test). f Nude mice intracranial tumor assay using wild type or S39A/T381A mutated YTHDF2 expressing GSC11 cells with YTHDF2 depletion. Brain sections stained with H&E show representative tumor xenografts. Tumor volumes were calculated using the formula V = ab2/2, where a and b are the tumor’s length and width, respectively. Data are mean ± S.D., n = 8 mice per group examined over two independent experiments (one-way ANOVA Tukey’s post hoc test). Source data are provided as a Source data file.
