Fig. 5: Interactions of 4E-T with eIF4E and PATL2 are required for its meiotic function.

a Xenopus stage-VI oocytes were injected with water or mRNA encoding the indicated Flag-4E-T^TRIM variant. 18 h after injection, oocytes were co-injected with mRNA encoding Flag-TRIM21 and either 4E-T^Ab1 or unspecific control (Ctrl) antibodies. 22 h after the second injection, oocytes were lysed and immunoblotted as indicated. One representative experiment of three independent biological replicates is shown. b Oocytes from (a) (Ctrl TRIM + H₂O, n = 50 oocytes; 4E-T^Ab1 TRIM + H₂O, n = 51 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM, n = 51 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM Δ4Ec, n = 53 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM Δ4Enc, n = 53 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM ΔPATL, n = 54 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM ΔLSM14A, n = 59 oocytes) were treated with PG 22 h after the second injection. Percentage of oocytes undergoing GVBD in 390 min after PG addition and median time to GVBD are given as mean ± s.d. from three independent biological replicates. p values were calculated using one-way ANOVA with Dunnett’s multiple comparisons test. c TRIM-Away of 4E-T and expression of the indicated Flag-4E-T^TRIM variants was performed as described in (a). 22 h after the second injection, oocytes were lysed for immunoblotting as indicated. One representative experiment of three independent biological replicates is shown. d Oocytes from (c) (Ctrl TRIM + H₂O, n = 67 oocytes; 4E-T^Ab1 TRIM + H₂O, n = 64 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM, n = 63 oocytes; 4E-T^Ab1 TRIM + Flag-4E-T^TRIM Δ4Ec ΔPATL, n = 62 oocytes) were treated with PG 22 h after the second injection. Percentage of oocytes undergoing GVBD in 600 min after PG addition and median time to GVBD are given as mean ± s.d. from three independent biological replicates. p values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. A detailed analysis of the GVBD timing in the different conditions can be found in Fig. S6a. e Xenopus stage-VI oocytes were injected with mRNA encoding Flag-4E-T^TRIM or Flag-4E-T^TRIM Δ4Ec ΔPATL. 18 h after injection, oocytes were lysed and separated by sucrose density gradient centrifugation. Gradient fractions were analyzed by immunoblot as indicated. One representative experiment of three independent biological replicates is shown. f Xenopus stage-VI oocytes were injected with water or mRNA encoding the indicated Flag-4E-T^TRIM variant. 18 h after injection, oocytes were lysed and subjected to immunoprecipitation with Flag antibodies. Samples were immunoblotted as indicated. Asterisk indicates IgG HC. In parallel, RNA was isolated from the same samples and analyzed by qRT-PCR for the indicated mRNAs. Values in input and IP samples were normalized to the Flag-4E-T^TRIM conditions and are given as mean ± s.d. from three independent biological replicates. p values within input and IP conditions were calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Source data including additional loading controls are provided as a Source Data file.