Fig. 2

FMRP controls EP300 protein synthesis in NPCs of 6-month old mice. a, b Fmr1 KO 6-mo NPCs exhibited reduced proliferation compared to WT NPCs as assessed by incorporation of the thymidine analog, BrdU (a, red, BrdU; blue, DAPI. scale bar, 20 µm) and quantitative analysis (b, n = 3–4). c, d Fmr1 KO 6-mo NPCs differentiated into fewer neurons compared to WT NPCs as assessed by a neuronal marker Tuj1⁺ (c, red, BrdU; blue, DAPI. scale bar, 20 µm) followed by quantitative analysis (d, n = 3). e, f Western blot analyses of EP300 protein expression levels in WT and Fmr1 KO 6-mo NPCs, (n = 3). GAPDH was used as a loading control. g ELISA analyses of EP300 protein levels in WT and Fmr1 KO 6-mo NPCs (n = 3). h RNA-IP-qPCR analysis showing FMRP was associated with Ep300 mRNA in NPCs (n = 3). i Quantitative real-time PCR analyses showing no significant differences in Ep300 mRNA levels between WT and Fmr1 KO 6-mo NPCs (n = 3). j Workflow for detection and identification of newly synthesized proteins using BONCAT (bio-orthogonal non-canonical amino acid tagging). k Visualization of Biotin-tagged, newly synthesized proteins in WT and Fmr1 KO NPCs using Biotin blot. l, m Sample western blot (upper panel) and quantitative analysis (lower panel, n = 3) of newly synthesized EP300 protein in WT and Fmr1 KO NPCs as determined by using BONCAT. n–q Western blot analyses of acetylation of histone H3 (n, o) and H4 (p, q) in WT and Fmr1 KO 6-mo NPCs (n = 3). Total histone H3 or H4 was used as a loading control for acetylation of histones. r–t Western blot analyses of total histone H3 and H4 levels in WT and Fmr1 KO NPCs (n = 3). GAPDH was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001. n.s.: no significant difference. Student’s t-tests were used for data analyses. Data are presented as mean ± s.e.m.