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Fig. 7 | Genome Biology

Fig. 7

From: Control of immediate early gene expression by CPEB4-repressor complex-mediated mRNA degradation

Fig. 7

CPEB1-4 repressor complex formation by interaction with the CCR4-NOT complex. A Co-IP of endogenous NOT1, NOT2, NOT7, and NOT10 with ectopically expressed, SBP-tagged human CPEB1, CPEB3, CPEB4, and mouse (m) CPEB2, in HeLa cells. The IP was carried out using streptavidin beads in the absence and presence of 26 U RNase A/T1, and precipitated proteins were detected by western blot analysis using specific antibodies as indicated. The asterisk denotes a non-specific band. B Western blot analysis of HA-tagged human CPEB1, CPEB3, CPEB4, and mouse mCPEB2 fused to the bacteriophage PP7 coat protein (PP7cp) in HeLa cells. C For the tethering assay, HeLa cells were transiently transfected with HA-PP7cp, HA-PP7cp-CPEB1, -CPEB3, -CPEB4, or -mCPEB2 together with a β-globin reporter mRNA containing 6 repeats of the PP7bs. β-globin reporter mRNA decay and deadenylation were analyzed as in Fig. 3E. D Deadenylation was visualized by densitometric analysis of the β-globin mRNA signal. RNA digested with RNase H in presence of oligo-dT serves as a reference for fully deadenylated (poly(A)-) RNA. The signal intensity was plotted as a function of the poly(A) tail length and the maximum of the 4-h timepoint is indicated by a red dashed line. E Decay of β-globin mRNA in the tethering assay is depicted after normalization to 18S rRNA (mean values ± SD; n = 3). F Half-lives of the β-globin mRNA in the tethering assays were calculated following first-order decay kinetics (mean values ± SD; n = 3) and p-values were calculated using a two-sided, paired t-test; * p < 0.05. Note that the β-globin mRNA decay curve in E and the reporter mRNA half-life measured after transfection of HA-PP7cp-CPEB4 in F are the same as in Fig. 3F and G

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