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Fig. 1 | Genome Biology

Fig. 1

From: Comparative analyses of vertebrate CPEB proteins define two subfamilies with coordinated yet distinct functions in post-transcriptional gene regulation

Fig. 1

CPEBs are co-expressed and co-regulated by phosphorylation in the meiotic cell cycle. A Endogenous CPEB2 and CPEB3 immunoblots from b-isox-precipitated extracts from the indicated meiotic maturation and early development time points. CPEB2, n = 2; CPEB3, n = 4. Vinculin immunoblot was used as a loading control. The number of oocytes loaded is indicated in parentheses. B Table showing the number of peptide spectrum matches (PSMs) of the CPEBs detected by MS/MS after b-isox-precipitation of meiotic maturation lysates, n = 1. C, D. Western blots of HA-CPEB2 (n = 2) and HA-CPEB3 (n = 2) overexpressing oocytes during meiotic maturation. Vinculin immunoblot was used as a loading control. E Lambda phosphatase assays (λ-PPase) of HA-CPEB2 (n = 2) and HA-CPEB3 (n = 2) overexpressing oocytes at the indicated time points. Western blots of anti-HA and anti-vinculin are shown. F CPEB3 phospho-to-total occurrence ratios determined by MS/MS for the indicated residue positions on prophase I (PI) versus progesterone-treated oocytes (+ Prog.). The ratios were calculated from the pool of 4 biological replicates. Only positions with a relative gain of phosphorylation upon progesterone treatment are displayed. Proline-directed sites are highlighted in bold. Error bars represent the ratio error. G Relative positions of the 18 proline-directed sites in CPEB3. The NTD is white-shaded, whereas the CTD is gray-shaded. H Wild-type (wt) and phosphomimetic (DE) CPEB3-NTD [γ-32P]-ATP incorporation upon incubation with oocyte lysates at the indicated maturation time points (n = 2). I Western blot against HA-tag and endogenous CPEB1 of stage VI ( −) and progesterone stimulated oocytes ( +) overexpressing wild-type HA-CPEB3 (wt), phosphonull HA-CPEB3 (A), and phosphomimetic HA-CPEB3 (DE). Not injected (NI) (n = 6). J Relative positions of the 20 proline-directed sites in CPEB2. The NTD is white-shaded, whereas the CTD is gray-shaded. K [γ-32P]-ATP incorporation by wild-type (wt) or phosphomutant (DE) CPEB2-NTD upon incubation with oocyte lysates at the indicated maturation time points (n = 3). L Western blot against HA-tag and CPEB1 of stage VI ( −) and progesterone stimulated oocytes ( +) overexpressing wild-type HA-CPEB2 (wt), not injected (NI), phosphonull HA-CPEB2 (A), and phosphomimetic HA-CPEB2 (DE) (n = 3). M The mean inhibition of [γ-32P]-ATP incorporation to CPEB2-NTD, CPEB3-NTD, or Histone H1 (H1) by increasing inhibitor concentrations. Data points represent the mean and standard deviation (n ≥ 3). Abbreviations: PI, prophase-I; MI, metaphase-I; MII, metaphase-II; hpf, hours post-fertilization; b-isox, biotinylated-isoxazole; λ-PPase, lambda phosphatase; In, input; Prog. or P, progesterone; DE, phosphomimetic mutant; A, phosphonull mutant; NTD, N-terminal domain; NL, no-lysate; NI, not-injected

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