CK2 phosphorylates mammalian FMRP S499. CK2 phosphorylates mammalian FMRP S499. A, Immunoblots for 3-h treatment of N2a cells with vehicle (DMSO), D4476 (25 µm), IC261 (20 µm), PHA-767491 (5 µm), DRB (50 µm), TBB (25 µm), or βARK (200 µm) followed by Western blot for protein indicated on the left. B, Quantification of pFMRP/tFMRP signal in A. ns, not significant (Kruskal–Wallis one-way ANOVA analysis [H(8) = 4.56, p = 0.8034], n = 4). C, Immunoblot for 24-h treatment of N2a cells with the same agents listed in A. D, Quantification of pFMRP/tFMRP signal in C (Kruskal–Wallis one-way ANOVA [H(8) = 7.239, p = 0.5111], n = 4, error bars = SEM). E, Baseline, untreated N2a cells were collected at time 0, and the remainder of the cells were treated with either DMSO or CX-4945 (5 or 1 µm) for 24 h. tFMRP and pFMRP S499 signals increased in DMSO-treated samples; however, only tFMRP increased in CX-treated samples, thereby causing a significant reduction in relative FMRP S499 phosphorylation. All immunoblot signals are from the same membrane; however, intervening lanes have been removed for clarity. F, Quantification of pFMRP S499 to tFMRP ratio from D; one-way ANOVA, n = 4, error bars = SEM, *p < 0.05. G, HEK293 cells were collected at baseline (time 0) or treated for 24 h with DMSO or CX-4945. H, CX-4945 significantly reduced FMRP S499 phosphorylation compared with DMSO (one-tailed Mann–Whitney test, p = 0.0143). I, J, Mouse cortical neurons at 7 d in vitro treated with 1 µm CX-4945 for 24 h exhibited a significant reduction in FMRP S499 phosphorylation compared with DMSO-treated neurons (one-tailed Mann–Whitney test, p = 0.05). Christopher M. Bartley et al. eNeuro 2016;3:ENEURO.0092-16.2016 ©2016 by Society for Neuroscience