FMRP S499 is phosphorylated by multiple kinases in vitro.

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FMRP S499 is phosphorylated by multiple kinases in vitro. FMRP S499 is phosphorylated by multiple kinases in vitro. A, FMRP S499 phosphorylation is not altered by 3-h staurosporine treatment. N2a cells were treated for 3 h with increasing doses of staurosporine. Staurosporine decreased rpS6 S240/244 phosphorylation without affecting FMRP S499 phosphorylation. B, Quantification of rpS6 S240/244 (pS6/S6, blue) and FMRP S499 phosphorylation after staurosporine treatment. There was no significant effect of staurosporine treatment on FMRP phosphorylation (Kruskal–Wallis one-way ANOVA [H(5) = 2.32, p = 0.8034]), but there was a significant effect on rpS6 S240/244 phosphorylation (Kruskal–Wallis one-way ANOVA [H(5) = 16.04, p = 0.0067]. Post hoc Dunn’s test for multiple comparisons showed p < 0.05 for 50- and 150-µm treatment groups compared with 0 µm. n = 3, error bars = SEM. C, Initial Kinexus kinase assay. rFRMP S500 was incubated with recombinant kinase listed above the SDS-PAGE on 10% Tris-glycine gels. Samples were run on two independent blots. The leftmost lane is the ladder. Arrows indicate where intervening lanes have been removed for clarity. D, Repeat kinase assay including GRK2. The two panels are from the same membrane. After transfer, the membrane was probed with anti-pFMRP S499, stripped, and reprobed with tFMRP. Christopher M. Bartley et al. eNeuro 2016;3:ENEURO.0092-16.2016 ©2016 by Society for Neuroscience