Fig. 9
CFP1 function is inhibited by phosphorylation. a Western blots showing levels of HA-tagged CFP1 (WT and mutants) and H3K4me3 in microinjected oocytes, at 8 h after release into meiotic maturation. Phosphorylated ERK1/2 was blotted as a marker of meiotic resumption, and α-tubulin was blotted as a loading control. Fully grown GV oocytes from WT mice were used for mRNA microinjection throughout the experiments presented in this figure. b Ratio of H3K4me3 levels between MI and GV stages in oocytes overexpressing WT and mutated CFP1, based on western blot results in (a, c). c Western blots showing H3K4me3 levels in oocytes overexpressing indicated CFP1 mutants, at 8 h after release into meiotic maturation. d Western blot results showing H3K4me3 levels in GV oocytes overexpressing the indicated CFP1 mutants for 12 h after mRNA microinjection. e Co-IP results showing interaction between CFP1 and histone H3.3 in HeLa cells transfected with plasmids expressing the indicated recombinant proteins. f Expression levels of WT and phosphorylation site-mutated CFP1 in GV oocytes at 12 h after mRNA microinjection. g Immunofluorescence results showing CFP1 localization and H3K4me3 levels in the nuclei of GV oocytes after mRNA microinjection as in (d). Scale bar, 5 μm. Nucleoli are indicated by asterisks. h, i Quantification of H3K4me3 signals in g, j, respectively. Error bars, S.E.M. ***P < 0.001 by two-tailed Student’s t tests. n.s., non-significant. j Immunofluorescence on chromatin spread made from GV oocytes after mRNA microinjection as in g, showing levels of chromatin-bound CFP1. Scale bar, 5 μm
