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. 2019 Nov 20;8:e48718. doi: 10.7554/eLife.48718

Figure 4. EphrinA1 co-stimulation blocks Netrin-1 induced receptor-ribosome dissociation and selective translation.

(A) Relative PLA quantification of DCC and RPL5/uL18 compared to control after Netrin-1, EphrinA1, or co-stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; **p<0.01). (B, C) Puromycin QIF relative to control after Netrin-1, EphrinA1 or co-stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; ***p<0.0001). (D) Relative mRNA quantification after DCC IP of hnrnph1 and ctnnb1 mRNA (unpaired t-test with Welch’s corrections on dCT values; three biological replicates; bars indicate mean, error bars indicate SEM; *p=0.02 for hnrnph1; **p=0.0018 for ctnnb1). (E, F) β-Catenin QIF relative to control after Netrin-1, EphrinA1, Sema3A or Netrin-1 and EphrinA1 co-stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; ***p<0.0001). (G, H) hnRNPH1 QIF relative to control after Netrin-1, EphrinA1, Sema3A or Netrin-1 and EphrinA1 co-stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; ***p<0.0001; *p=0.0164). Scale bars, 5 μm. For all QIF experiments, numbers in bars indicate amount of growth cones quantified collected from at least three independent experiments.

Figure 4—source data 1. Spreadsheet containing all normalized PLA count values for each axonal growth cone in Figure 4A, all normalized puromycin intensity values for each axonal growth cone in Figure 4C, all normalized ß-Catenin intensity values for each axonal growth cone in Figure 4F and all normalized hnRNPH1 intensity values for each axonal growth cone in Figure 4H.

Figure 4.

Figure 4—figure supplement 1. EphrinA1 co-stimulation blocks Netrin-1 induced receptor-ribosome dissociation and selective translation of rps14.

Figure 4—figure supplement 1.

(A) Relative PLA quantification of Nrp1 and RPS23/uS12 compared to control after Sema3A, EphrinA1, or co-stimulation with Sema3A and EphrinA1 (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; *p=0.032078; **p<0.018577; ***p<0.001). (B) pERK1/2 QIF relative to control after Netrin-1, EphrinA1 or Netrin-1 and EphrinA1 co-stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; ***p<0.0001). (C) Relative mRNA quantification after DCC IP of rps14 mRNA (unpaired t-test with Welch’s corrections on dCT values; three biological replicates; bars indicate mean, error bars indicate SEM; ***p=0.0003). (D) RPS14 QIF relative to control after Netrin-1, EphrinA1 or Netrin-1 and EphrinA1 co-stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; ***p<0.0001; *p=0.026544). (E) RpsRPS14 QIF relative to control after Netrin-1 or Sema3A stimulation (one-way ANOVA with Bonferroni’s multiple comparisons test; bars indicate mean, error bars indicate SEM; ***p<0.0001; *p<0.05). Scale bars, 5 μm. For all QIF experiments the numbers in bars indicate amount of growth cones quantified collected from three independent experiments.
Figure 4—figure supplement 1—source data 1. Spreadsheet containing all normalized PLA count values for each axonal growth cone in Figure 4—figure supplement 1A, all normalized pERK1/2 intensity values for each axonal growth cone in Figure 4—figure supplement 1B and all normalized RPS14 intensity values for each axonal growth cone in Figure 4—figure supplement 1D and E.