Journal: PLoS ONE

Article Title: Plasma protein profiling reveals candidate biomarkers for multiple sclerosis treatment

doi: 10.1371/journal.pone.0217208

Figure Lengend Snippet: The target proteins that changed significantly during natalizumab treatment.

Article Snippet: For the RTN3 antibody (HPA015649, Atlas antibodies), wells were coated with two RTN3 protein fragments covering different parts of the protein (Figure A in ) that were produced in our lab (materials and methods in ); the amino acids sequence from 2–299 (RTN3_A) (Figure B in ) and the amino acids sequence from 254–700 (RTN3_B).

Techniques:

Journal: PLoS ONE

Article Title: Plasma protein profiling reveals candidate biomarkers for multiple sclerosis treatment

doi: 10.1371/journal.pone.0217208

Figure Lengend Snippet: a) changes in PEBP1 in the screening cohort. b) changes in RTN3 in the screening cohort. c) changes in PEBP1 in the replication cohort. d) changes in RTN3 in the replication cohort. The Y axis represents the mean prediction of the change of PEBP1 and RTN3 levels during treatment obtained from the linear mixed model and the error bars indicate the standard error of the mean. *p value from the linear mixed model.

Article Snippet: For the RTN3 antibody (HPA015649, Atlas antibodies), wells were coated with two RTN3 protein fragments covering different parts of the protein (Figure A in ) that were produced in our lab (materials and methods in ); the amino acids sequence from 2–299 (RTN3_A) (Figure B in ) and the amino acids sequence from 254–700 (RTN3_B).

Techniques:

Journal: PLoS ONE

Article Title: Plasma protein profiling reveals candidate biomarkers for multiple sclerosis treatment

doi: 10.1371/journal.pone.0217208

Figure Lengend Snippet: Comparison of PEBP1 and RTN3 between HCs and MS patients at baseline and 12 month natalizumab treatment. Mean (standard error of the mean).

Article Snippet: For the RTN3 antibody (HPA015649, Atlas antibodies), wells were coated with two RTN3 protein fragments covering different parts of the protein (Figure A in ) that were produced in our lab (materials and methods in ); the amino acids sequence from 2–299 (RTN3_A) (Figure B in ) and the amino acids sequence from 254–700 (RTN3_B).

Techniques: Comparison

Journal: PLoS ONE

Article Title: Plasma protein profiling reveals candidate biomarkers for multiple sclerosis treatment

doi: 10.1371/journal.pone.0217208

Figure Lengend Snippet: The changes in a) PEBP1 and b) RTN3 plasma levels and MS severity. Moderate responders are 20 patients with the highest decrease in MSSS during treatment. Mild responders are 20 patients with the lowest decrease in MSSS during treatment. The Y axis represents the mean prediction of the change of PEBP1 and RTN3 levels during treatment obtained from the linear mixed model and the error bars indicate the standard error of the mean. *p value from the linear mixed model.

Article Snippet: For the RTN3 antibody (HPA015649, Atlas antibodies), wells were coated with two RTN3 protein fragments covering different parts of the protein (Figure A in ) that were produced in our lab (materials and methods in ); the amino acids sequence from 2–299 (RTN3_A) (Figure B in ) and the amino acids sequence from 254–700 (RTN3_B).

Techniques: Clinical Proteomics

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Diagram to show timeline of treatments and tissue harvesting. ( B ) Microarray data to show changes in RTN3 mRNA in DC, SN and pSN+DC lesions ( n = 8 DRG/group (4 rats/group)). ( C ) qRT-PCR to confirm upregulated RTN3 mRNA in SN and pSN+DC injury models ( n = 16 DRG/group (8 rats/group)). ( D ) Western blot to show levels of levels of RTN3 in control, DC, SN and pSN+DC injury models ( n = 16 DRG/group (8 rats/group)). ( E ) Densitometry to confirm upregulation of RTN3 in SN and pSN+DC injury models ( n = 16 DRG/group (8 rats/group)). ( F ) Immunohistochemistry to show immunolocalization of RTN3 (green) in DRGN (red). Data are means ± SEM. Scale bars in (F) = 50µm. **P = 0.001; ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Microarray, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Timeline of experiment and analysis methods. ( B ) Western blot to show RTN3 plasmids overexpress RTN3 protein. ( C ) Densitometry to show that RTN3 was significantly upregulated in RTN3 transfected cultures. ( D ) Representative images to show DRGN neurite outgrowth after RTN3 overexpression. RTN3 overexpression increases ( E ) the mean DRGN neurite length, ( F ) % DRGN with neurites but does no affect ( G ) DRGN survival. LF2000 = Lipofectamine 2000, AU = arbitrary units, *** P =0.0001, one-way ANOVA with Dunnett’s post hoc test, n = 9 wells/treatment. Scale bars in (D) = 100µm.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Western Blot, Transfection, Over Expression

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Experimental timeline for analysis of RTN3 knockdown in preconditioned DRGN. ( B ) qRT-PCR to show ∼80% knockdown of RTN3 using siRTN3. ( C ) Western blot to show levels of RTN3 protein after shRNA-mediated knockdown in DRGN. β-actin is used as a loading control. ( D ) Densitometry to show that PEI-shRTN3 significantly knocks down RTN3 protein in DRGN cultures. ( E ) Representative images to show neurite outgrowth after LF2000, siEGFP and siRTN3 treatment. ( F ) Quantification to show the mean longest neurite length after shRNA-mediated knockdown of RTN3. ( G ) Quantification to show % DRGN with neurites after shRNA-mediated knockdown of RTN3. ( H ) Quantification to show that DRGN survival is unaffected after shRNA-mediated knockdown of RTN3. Data are means ± SEM. Scale bars in (E) = 100µm. ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test. n = 8 wells/condition.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Quantitative RT-PCR, Western Blot, shRNA

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: We determined that 2µg of shRTN3 plasmids were required for maximal RTN3 knockdown. Data are means ± SEM. n = 9 wells/treatment. ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques:

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Timeline for experimental analysis of RTN3 overexpression in preconditioned DRGN. ( B ) Fold-change in mRNA after RTN3 overexpression in preconditioned DRGN. ( C ) Western blot to show overexpressed levels of RTN3 protein. β-actin is used as a protein loading control. ( D ) Densitometry to show significant RTN3 overexpression by PEI-RTN3. ( E ) Representative images to show neurite outgrowth after DC+PEI-EGFP, pSN+DC+PEI-EGFP and pSN+DC+PEI-RTN3 treatment. ( F ) Quantification to show the mean neurite length after DC+PEI-EGFP, pSN+DC+PEI-EGFP and pSN+DC+PEI-RTN3 treatment ( G ) Quantification to show % DRGN with neurites after DC+PEI-EGFP, pSN+DC+PEI-EGFP and pSN+DC+PEI-RTN3 treatment. Data are means ± SEM. ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test. Scale bars in (E) = 100µm. n = 9 wells/treatment.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Over Expression, Western Blot

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Immunostaining for RTN3 and βIII-tubulin in DRGN cultures, challenged with Aβ 1-42 to show both upregulation of RTN3 in DRGN cultures and normal neurite outgrowth. High power images to show normal appearance of neurites in ( B ) control and ( C ) RTN3 overexpressed DRGN. ( D ) The mean neurite length, ( E ) % DRGN with neurites and ( F ) DRGN survival are unaffected by Aβ 1-42 exposure. Scale bars in (A) = 50µm, scale bars in (B and C) = 200 µm. ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Timeline for experimental analysis of Protrudin and its interactors in RTN3-overexpressed DRGN. Representative western blots to confirm knockdown of ( B ) Protrudin, ( C ) FYCO1, ( D ) RAB7 and ( E ) SYT7. β-actin is used as a protein loading control. Representative images to show neurite outgrowth in ( F ) RTN3 overexpressed DRGN controls and after knockdown of ( G ) Protrudin, ( H ) FYCO1, ( I ) RAB7 and ( J ) SYT7 in RTN3-overexpressed cultures. ( K ) Quantification to show attenuation of RTN3-overexpressed DRGN neurite outgrowth by shProtrudin, shFYCO1, shRAB7 and shSYT7. (L ) DRGN survival remains unaffected by knockdown of Protrudin, FYCO1, RAB7 and SYT7 in RTN3-overexpressed DRGN. n = 9 wells/condition Scale bars in (F-J) = 100µm. Data are means ± SEM. ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: qRT-PCR shows lack of activation of interferon-related or innate immunity-related genes after siRNA transfections in RTN3-overexpressed DRGN. The positive control sequence (sip75 NTR ; ) invokes 12-17-fold changes in all of these genes. Data are means ± SEM. n = 6 wells/treatment.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Quantitative RT-PCR, Activation Assay, Transfection, Positive Control, Sequencing

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Representative immunohistochemistry to show RTN3 immunoreactivity in control (pSN+DC+PEI-EGFP) and after overexpression of RTN3 in pSN+DC+PEI-RTN3-treated animals. ( B ) Western blot to show levels of RTN3 protein after overexpression of RTN3. β-actin is used as a protein loading control. ( C ) Densitometry showed significant upregulation of RTN3 protein after overexpression using RTN3 plasmids. ( D ) qRT-PCR to show knockdown in protrudin mRNA over time after in vivo DRG injection PEI-shProtrudin. Data are means ± SEM. n = 18 rats/group/test. Scale bars in (A) = 50µm. ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Immunohistochemistry, Over Expression, Western Blot, Quantitative RT-PCR, In Vivo, Injection

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Representative longitudinal sections of the spinal cord immunostained with GAP43 antibodies after treatment with pSN+DC+PEI-EGFP, pSN+DC+PEI-RTN3+shEGFP and pSN+DC+PEI-RTN3+shProtrudin (* = lesion site) (C to R = caudal to rostral). FluoroRuby (FR) traced axons in the same sections as pSN+DC+PEI-RTN3+shEGFP confirms overlap of GAP43 + axons (arrowheads), unequivocally demonstrating axon regeneration. Inset (i) shows high power images of GAP43 + axons in the boxed region (i). Inset (ii) and (iii) show high power images of FR + axons in boxed regions (ii) and (iii). Note the overlap (arrowheads) between GAP43 + axons in inset (i) and FR + axons in inset (ii). ( B ) Quantification of the % of GAP43 + axons at different distances caudal and rostral to the lesion site. Scale bars in (A) = 200µm. n = 18 rats/group. *P = 0.05, **P = 0.001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques:

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Representative Spike 2 processed CAP traces in sham, DC+PEI-EGFP, pSN+DC+PEI-EGFP, pSN+DC+PEI-RTN3, pSN+DC+PEI-RTN3+shEGFP and pSN+DC+PEI-RTN3+shProtrudin-treated animals. ( B ) Negative CAP amplitudes at different stimulation intensities in sham, DC+PEI-EGFP, pSN+DC+PEI-EGFP, pSN+DC+PEI-RTN3, pSN+DC+PEI-RTN3+shEGFP and pSN+DC+PEI-RTN3+shProtrudin-treated animals. ( E ) CAP areas at all stimulation intensities in sham, DC+PEI-EGFP, pSN+DC+PEI-EGFP, pSN+DC+PEI-RTN3, pSN+DC+PEI-RTN3+shEGFP and pSN+DC+PEI-RTN3+shProtrudin-treated animals. ( F ) Mean tape sensing and removal times in sham, DC+PEI-EGFP, pSN+DC+PEI-EGFP, pSN+DC+PEI-RTN3, pSN+DC+PEI-RTN3+shEGFP and pSN+DC+PEI-RTN3+shProtrudin-treated animals. ( G ) Mean error ratio to show the number of slips over the total number of steps in sham, DC+PEI-EGFP, pSN+DC+PEI-EGFP, pSN+DC+PEI-RTN3, pSN+DC+PEI-RTN3+shEGFP and pSN+DC+PEI-RTN3+shProtrudin-treated animals. Data are means ± SEM. n = 18 rats/group. ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test. # P = 0.0012, linear mixed models (LMM); ## P = 0.00012, generalised linear mixed models (GLMM).

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques:

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Immunohistochemistry to show induction of GAP43 + immunoreactivity in RTN3 overexpressed DRGN (as measured by the reporter gene EGFP in green; arrows). ( B ) qRT-PCR to demonstrate upregulation of RAGs ( gap43, sprr1a, cebp-epsilon, atf3 and galanin ) after preconditioning lesions and further upregulation after RTN3 overexpression. Data are means ± SEM. n = 12 rats/group/test. Scale bars in (A) = 25µm. ***P = 0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Immunohistochemistry, Quantitative RT-PCR, Over Expression

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Western blot to show that RTN3 is significantly overexpressed in retinal cells treated with RTN3 plasmids. ( B ) Densitometry shows greater than 3-fold upregulation of RTN3 protein levels in retinal cultures by RTN3 plasmids. ( C ) Representative images stained with βIII-tubulin and ( D ) quantification of the mean neurite length and ( E ) % RGC with neurites to show that RTN3 overexpression increased neurite length and the number of RGC with neurites, an effect which was ablated after knockdown of Protrudin. ( F ) RTN3 overexpression promotes significant RGC survival, which was not affected by protrudin knockdown. Data are means ± SEM. n = 9 wells/treatment. Scale bars in (C) = 100µm; ***P=0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Western Blot, Staining, Over Expression

Journal: bioRxiv

Article Title: Overexpression of Reticulon 3 enhances CNS axon regeneration and functional recovery after injury

doi: 10.1101/2021.05.24.445420

Figure Lengend Snippet: ( A ) Western blot and ( B ) quantification to show that RTN3 plasmids significantly upregulate RTN3 protein in the retina after intravitreal injection in vivo . ( C ) Immunohistochemistry to localise RTN3 protein to the RGCs (green) in the ganglion cell layer (GCL) and to the inner plexiform and inner nuclear layer (INL). ONL = outer nuclear layer. ( D ) High power images to show RTN3 protein localised to βIII-tubulin + RGCs (arrows) in the GCL. ( E ) GAP43 immunohistochemistry and ( F ) quantification to show that in control optic nerve, few if any GAP43 + axon pass beyond the lesions site (*) whilst in RTN3 overexpressed eyes, significant GAP43 + axons are present beyond the lesion site, an effect which is obliterated when Protrudin is knocked out at the same time as RTN3 overexpression. ( G ) Representative images to show FluorGold (FG) backfilled RGC in retinal wholemounts after RTN3 overexpression. ( H ) Quantification of the number of FG + RGC in retinal wholemounts shows that overexpression of RTN3 is significantly neuroprotective, an effect which is independent of protrudin. ( I ) Representative ERG traces and ( J ) quantification of the pSTR amplitude shows significant improvements in RTN3 overexpressed eyes which is ablated after protrudin knockdown. Arrows show peak of pSTR. Data are means ± SEM. n = 12 eyes/optic nerves/treatment. Scale bars in (C) = 100µm, scale bars in (D) = 25µm, scale bars in (E) = 200µm, scale bars in G = 50µm. ***P=0.0001, one-way ANOVA with Dunnett’s post hoc test.

Article Snippet: The following antibodies were used for immunoassays: primary antibodies - rabbit polyclonal anti-RTN3 (#ABN1723, Merck Millipore; 1:400 dilution), monoclonal anti-NF200 (#N5389, Sigma, 1:400), monoclonal anti-βIII-tubulin (#T8578, Sigma; 1:200), mouse anti-GAP43 (#33-5000 (clone 7B10), Invitrogen; 1:400); secondary antibodies - Alexa488 anti-rabbit (#A323731, Invitrogen; 1:400 dilution), Texas Red anti-mouse (#PA1-28626, Invitrogen; 1:400 dilution), Alexa488 anti-mouse (#A32723, Invitrogen; 1:400 dilution).

Techniques: Western Blot, Injection, In Vivo, Immunohistochemistry, Over Expression