Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A) Volcano plots showing statistically enriched proteins in DCC-IP and Nrp1-IP samples identified by permutation-based FDR-corrected t-test based on three biological replicates. The LFQ intensity of the DCC or Nrp1 pulldowns over IgG pulldowns are plotted against the - log10 p-value. FDR <0.05; S0 = 2. (B) Gene enrichment analysis of statistically enriched proteins in the DCC and Nrp1 pulldown samples. (C-F) Western blot validation of RP co-immunoprecipitation with DCC, Nrp1 and Robo2 but not with EphB2. (G-J) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldown or receptors pulldowns shows enrichment of rRNA in DCC, Nrp1, and Robo2 but not EphB2 pulldowns (unpaired two-tailed t-test; three biological replicates). Error bars indicate standard deviation; * p<0.05.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Western Blot, Immunoprecipitation, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A-C) Western blot validation of RP co-immunoprecipitation with DCC, Nrp1 and Robo2 in SH-SY5Y cells. (D-E) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldowns or receptor pulldowns shows enrichment of rRNA in DCC and Nrp1 IPs in SH-SY5Y cells (unpaired two-tailed t-test; three biological replicates). (F) iBAQ-based relative quantification of 60S or (G) 40S subunit RPs between DCC and Nrp1 (unpaired two-tailed t-test; three biological replicates). Error bars indicate standard deviation. *p<0.05.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Western Blot, Immunoprecipitation, Two Tailed Test, Standard Deviation

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A) Relative 18S and 28S ribosomal RNA abundance after control (IgG) pulldown or receptors pulldowns with or without EDTA or RNase A/T1 treatments (two-way ANOVA with Bonferroni’s multiple comparisons test; three biological replicates; Error bars indicate standard deviation; ***p<0.0001). (B) Western blot analysis and quantification of ribosomal proteins after DCC and (C) Nrp1 pulldowns. (two-way ANOVA with Bonferroni’s multiple comparisons test; three biological replicates; Error bars indicate standard deviation; **p<0.01; ***p<0.0001). (D, E) Volcano plots indicating both DCC and Nrp1 bind significantly to different RBPs (orange dots). FDR <0.05; S0 = 2. (F) DCC and Nrp1 each bind to differentially to the RBPs Staufen1 and hnRNPA2B1. FDR <0.05. (G) Distance matrix showing a high correlation between replicates and a distinct signature between samples. (H) Volcano plot showing differential expression analysis for DCC and Nrp1 pulldowns. (I) Enrichment analysis plot of known RBP targets detected in RNA-sequencing data after DCC and Nrp1 pulldown.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Standard Deviation, Western Blot, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A) Expansion imaging shows partial colocalization of DCC and (B) Nrp1 with ribosomal proteins (Scale bars, 5 μm). (C) Representative proximity ligation assay signal in axonal growth cones between DCC and RPL5/uL18, RPS4X/eS4 or IgG control (Scale bars, 5 μm). (D) Representative proximity ligation assay signal in axonal growth cones between Nrp1 and RPS3A/eS1, RPS23/uS12 or IgG control (Scale bars, 5 μm). (E) EphB2 and RPL5/uL18 show a significantly lower amount of PLA signal in axonal growth cones compared to DCC-RPL5/uL18 or Nrp1-RPS23/uS12 (Mann-Whitney test; ***p<0.0001; Scale bars, 5 μm). (F, G) Quantification of PLA signal in cue-stimulated axonal growth cones relative to control (unpaired two-tailed t-test; error bars indicate SEM; ***p<0.0001; *p = 0.0423). (H) 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; error bars indicate SEM; **p = 0.0058). For all PLA experiments, numbers in bars indicate amount of growth cones quantified collected from at least three independent experiments.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Imaging, Proximity Ligation Assay, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons

doi: 10.1101/673798

Figure Lengend Snippet: (A) PLA images showing DCC and RPL10A/uL1 are in close proximity in axonal growth cones, whereas DCC and IgG control generates little to no PLA signal. Scale bars, 5 μm. (B) Sema3A stimulation at protein-synthesis independent concentration does not decrease PLA signal between Nrp1 and RPS3A/eS1 (Mann-Whitney test; error bars indicate SEM; p = 0.2555). or (C) puromycin levels in axonal growth cones (Mann-Whitney test; error bars indicate SEM; p = 0.2487). (D) 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; Error bars indicate SEM; *p=0.032078; **p<0.018577; *** p<0.001). For all PLA and QIF experiments, numbers in bars indicate amount of growth cones quantified collected from at least three independent experiments.

Article Snippet: The following antibodies were used: mouse-anti-DCC (BD Biosciences, 554223); rabbit-anti-Nrp1 (Abcam, ab81321); goat-anti-Robo2 (R&D systems, AF3147); mouse-anti-EphB2 (Santa Cruz, sc130068) or an isotype control: rabbit IgG (Abcam, ab37415); mouse IgG1 (R&D systems, 11); mouse IgG2b (R&D systems, MAB004); goat IgG (R&D systems, AB-108-C).

Techniques: Concentration Assay, MANN-WHITNEY