Fig. 4: 14-3-3 strengthens the association between the SCMC and CDC25B. | Nature Communications

Fig. 4: 14-3-3 strengthens the association between the SCMC and CDC25B.

From: The subcortical maternal complex modulates the cell cycle during early mammalian embryogenesis via 14-3-3

Fig. 4

a Representative 5-ethynyl-2’-deoxyuridine (EdU) staining of in vitro fertilization (IVF) embryos derived from normal control and FlopedNull mouse zygotes. Mature eggs from control or FlopedNull female mice were incubated with normal sperms for 4 h and then were cultured in KSOM medium. The end of IVF were defined as time 0 h, and the presumptive fertilized eggs were treated with 10 μM EdU for denoted time intervals, fixed, permeabilized, and subjected to click reaction. Eggs used for IVF were harbored from 15 normal female mice (8 and 7 for two independent experiments) and 13 FlopedNull female mice (7 and 6 for two independent experiments). DNA was visualized with Hoechst 33342. Scale bar, 10 μm. DIC, differential interference contrast. b The in situ interaction between TLE6 and CDC25B in germinal vesicle (GV) oocytes, mature eggs, and zygotes from normal females were determined by proximity ligation assay (PLA) using antibodies to CDC25B and TLE6 or IgG for isotype control. DNA was visualized with DAPI. Scale bar, 10 μm. c Mouse GV oocytes were cultured for 5–7 h after co-microinjection with mCherry-Trim21 mRNA + isotype IgG (for control), or mCherry-Trim21 mRNA + anti-pan 14-3-3 antibody. The in situ interaction of TLE6 and CDC25B were determined by PLA using antibodies to CDC25B and TLE6 or IgG for isotype control. DNA was visualized with DAPI. Scale bar, 10 μm. d HEK-293F cells were co-transfected with expression vectors harboring Flag-tagged MATER (or a blank vector for negative control), Strep-tagged FLOPED, HA-tagged CDC25B and either Strep-tagged TLE6, TLE6S139A mutant, TLE6S209A mutant, or TLE6S139/209A double-site mutant as denoted. 60 h after transfection, cell lysates before (Input) or after immunoprecipitation with anti-Flag affinity agarose gel were immunoblotted as denoted. e HEK-293F cells were co-transfected with expression vectors as denoted. 60 h after transfection, cell lysates before (Input) or after immunoprecipitation with anti-Flag affinity agarose gel were immunoblotted for HA-tagged CDC25B, HA-tagged 14-3-3γ, Flag-tagged MATER, Strep-tagged FLOPED, and Strep-tagged TLE6.

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