Journal: PLoS ONE

Article Title: AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

doi: 10.1371/journal.pone.0061878

Figure Lengend Snippet: Microarray analysis to show fold-differences in mRNA levels compared to intact controls of described genes in rat DRG 1 d after DC lesion.

Article Snippet: Primary antibodies included: goat anti-human AMIGO, AMIGO2, AMIGO3 and TROY (all used at 1∶100 dilution and from Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-NGFR (1∶200 dilution; Sigma); rabbit anti-NGFR1 (1∶200 dilution; Alpha Diagnostics, San Antonio, TX, USA); and rabbit anti-LINGO1 (1∶400 dilution; Abcam, Cambridge, UK).

Techniques: Microarray

Journal: PLoS ONE

Article Title: AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

doi: 10.1371/journal.pone.0061878

Figure Lengend Snippet: ( A ) Semi-quantitative RT-PCR and ( B ) densitometry to show changes in mRNA levels of AMIGO isoforms and LINGO-1 expressed in non-regenerating DRGN compared to intact controls. GAPDH is used as a housekeeping gene. ( C ) Representative western blots and ( D ) densitometry to show changes in protein levels of AMIGO isoforms and LINGO-1 in non-regenerating DRGN relative to intact controls. ( E–H ) Immunohistochemistry in sections from intact controls and 1 d DC injured DRGN shows immunolabelling for AMIGO ( E ), AMIGO2 ( F ), AMIGO3 ( G ) and ( H ) LINGO-1. Insets in E-H show images from blocking peptide controls to demonstrate specificity of the relevant antibody Scale bars in E–H = 100 µm. *** = P<0.0001, ANOVA.

Article Snippet: Primary antibodies included: goat anti-human AMIGO, AMIGO2, AMIGO3 and TROY (all used at 1∶100 dilution and from Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-NGFR (1∶200 dilution; Sigma); rabbit anti-NGFR1 (1∶200 dilution; Alpha Diagnostics, San Antonio, TX, USA); and rabbit anti-LINGO1 (1∶400 dilution; Abcam, Cambridge, UK).

Techniques: Quantitative RT-PCR, Western Blot, Immunohistochemistry, Blocking Assay

Journal: PLoS ONE

Article Title: AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

doi: 10.1371/journal.pone.0061878

Figure Lengend Snippet: Microarray analysis to show fold-differences in mRNA levels of described genes compared to intact controls in rat DRG 10 d after DC lesion, sciatic nerve (SN) or pre-conditioning (pSN+DC) lesions.

Article Snippet: Primary antibodies included: goat anti-human AMIGO, AMIGO2, AMIGO3 and TROY (all used at 1∶100 dilution and from Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-NGFR (1∶200 dilution; Sigma); rabbit anti-NGFR1 (1∶200 dilution; Alpha Diagnostics, San Antonio, TX, USA); and rabbit anti-LINGO1 (1∶400 dilution; Abcam, Cambridge, UK).

Techniques: Microarray

Journal: PLoS ONE

Article Title: AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

doi: 10.1371/journal.pone.0061878

Figure Lengend Snippet: ( A ) Representative AMIGO3 localisation in DRGN (arrows = AMIGO3 + , arrowhead = AMIGO3 − ) and ( B ) quantification of the percentage ± SEM of DRGN which are AMIGO3 + and AMIGO3 − , in DRG cultures. ( C ) Western blotting and densitometry showing the levels of knockdown, compared to Scr-siAMIGO3 and siGFP after siAMIGO3 treatment. Re-stripped western blots are also shown for AMIGO, AMIGO2 and LINGO-1 to demonstrate that knockdown of AMIGO3 does not affect the levels of these molecules in culture. β-actin is used as a loading control. ( D ) Rho activation assay in untransfected and siRNA transfected DRG cells in the presence of CME. Total Rho levels were also detected by western blotting. Scale bar in (A) = 50 µm.

Article Snippet: Primary antibodies included: goat anti-human AMIGO, AMIGO2, AMIGO3 and TROY (all used at 1∶100 dilution and from Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-NGFR (1∶200 dilution; Sigma); rabbit anti-NGFR1 (1∶200 dilution; Alpha Diagnostics, San Antonio, TX, USA); and rabbit anti-LINGO1 (1∶400 dilution; Abcam, Cambridge, UK).

Techniques: Western Blot, Knockdown, Control, Activation Assay, Transfection