Journal: The Journal of Biological Chemistry

Article Title: Identification and characterization of Nanobodies targeting the EphA4 receptor

doi: 10.1074/jbc.M116.774141

Figure Lengend Snippet: Nb 39 and Nb 53 do not completely inhibit ephrin-A5 binding to EphA7. With Alphascreen technology we determined whether Nb 39 and Nb 53 could inhibit ephrin-A5 binding to EphA7. These Nbs did not completely inhibit the interaction between ephrin-A5 and EphA7 at the highest concentrations tested. This assay was done in triplicate, and data are represented as the mean ± S.D.

Article Snippet: To test the inhibition of EphA7 and ephrin-A5 interaction, His-tagged human ephrin-A5 (Sino Biological, Beijing, China) was biotinylated with a five-times molar excess of EZ link NHS biotin (Thermo Fisher Scientific) as is described for the Nbs.

Techniques: Binding Assay, Amplified Luminescent Proximity Homogenous Assay

Journal: Acta Neuropathologica Communications

Article Title: Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

doi: 10.1186/s40478-019-0759-6

Figure Lengend Snippet: EfnA5 gene expression levels are reduced in SOD1 G93A mice whereas efnA5 protein is not altered in ALS patients CSF. a EfnA5 gene expression was determined in the lumbar spinal cord of SOD1 WT and SOD1 G93A overexpressing mice ( N = 4–9). Data represents mean ± SEM, and different conditions were compared with a two-way ANOVA with Tukey’s multiple comparisons test: ** P < 0.01 as compared with SOD1 WT . b EfnA5 protein relative quantification in the CSF of patients carrying an ALS genetic known mutation (gALS) and patients without any known mutation (sALS) were compared to the levels found in the CSF of young and aged controls (Con) and asymptomatic ALS patients carrying a known mutation for the disease (asympt ALS). Boxes show median and interquartile range, and whiskers are minimum and maximum. The light-to-heavy (L/H) peptide ratio was used as relative quantification of EfnA5 protein

Article Snippet: For multiple reaction monitoring (MRM) analysis of efnA5 in CSF samples, 200 μL of CSF were spiked with TEAB buffer and a quantitative protein epitope signature tag (QPrEST, Atlas Antibodies AB, #QPrEST25042) of efnA5 as internal standard.

Techniques: Expressing, Mutagenesis

Journal: Acta Neuropathologica Communications

Article Title: Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

doi: 10.1186/s40478-019-0759-6

Figure Lengend Snippet: In the ventral horn of the spinal cord EfnA5 is mainly expressed in neurons. RNAscope in situ hybridization was performed to determine cell type-specific expression of EfnA5 . A total of 15–18 images of different lumbar spinal cord ventral horns from 3 different mice were analysed for every condition. a Representative images show lumbar spinal cord ventral horns of SOD1 WT and SOD1 G93A mice at 135 days of age that were stained with probes against EfnA5, Syp and Slc1a3 . Hoechst was used as a counter stain for nuclei. Scale bar = 50 μm. b Percentage of neurons ( Syp + cells) that stained positive for EfnA5 were quantified and shown as percentage of the total Syp + cells. c Glial cells ( Syp - cells) and d astrocytes ( Syp -, Slc1a3 + cells) positive for EfnA5 were quantified and represented as percentage of the total amount of glial cells. Single-cell EfnA5 expression was quantified by measuring EfnA5 puncta density (dots/μm 2 ) in e neurons, f glial cells and g astrocytes. Data represents mean ± SEM, and different conditions were compared with a two-tailed t-test: * P < 0.05; ** P < 0.01 as compared to SOD1 WT . b and e Data represents mean ± SEM, and different conditions were compared with a two-way ANOVA with Tukey’s multiple comparisons test: * P < 0.05 as compared with SOD1 WT ; ## P < 0.01 as compared with 150–400 μm 2 neurons

Article Snippet: For multiple reaction monitoring (MRM) analysis of efnA5 in CSF samples, 200 μL of CSF were spiked with TEAB buffer and a quantitative protein epitope signature tag (QPrEST, Atlas Antibodies AB, #QPrEST25042) of efnA5 as internal standard.

Techniques: In Situ Hybridization, Expressing, Staining, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

doi: 10.1186/s40478-019-0759-6

Figure Lengend Snippet: Reduction of efnA5 levels in SOD1 G93A mice is detrimental and shortens survival and disease duration. Disease progression was closely monitored in a - f SOD1 G93A EfnA5 +/+ and SOD1 G93A EfnA5 +/− mice from the age of 60 days onwards ( N = 26–28). a Weight and b and c motor performance decline over time on b the hanging wire (HW) and c rotarod tests is shown. Data is represented as mean ± SEM and two-way ANOVA test with repeated measurements was used to determine differences among genotypes. d Disease onset differences were compared between the two genotypes with the Log-rank test whereas e survival was analysed with the two-tailed Mann-Whitney test for non-parametric data: * P < 0.05. f Disease duration was analysed with a two-tailed student t-test: * P < 0.05. g EfnA5 expression levels in the lumbar spinal cord of end-stage mice were determined by qPCR and data is represented as mean ± SEM ( N = 7–9). Different genotypes were compared with a one-way ANOVA with Tukey’s multiple comparisons test: *** P < 0.001

Article Snippet: For multiple reaction monitoring (MRM) analysis of efnA5 in CSF samples, 200 μL of CSF were spiked with TEAB buffer and a quantitative protein epitope signature tag (QPrEST, Atlas Antibodies AB, #QPrEST25042) of efnA5 as internal standard.

Techniques: Two Tailed Test, MANN-WHITNEY, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

doi: 10.1186/s40478-019-0759-6

Figure Lengend Snippet: Electromyography decline, neuronal loss and neuromuscular junction denervation in SOD1 G93A mice is not affected in SOD1 G93A mice with reduced efnA5 levels. a Compound muscle action potential amplitude (expressed in mV) was measured in SOD1 G93A EfnA5 +/+ and SOD1 G93A EfnA5 +/− mice from the age of 60 days (d) onwards. Data is represented as as mean ± SEM ( N = 7–8). b The number of motor neurons of different soma area was counted in the ventral horn of the lumbar spinal cord. Data is represented as mean ± SEM ( N = 6–7) and it was analysed with a two-way ANOVA. Representative image shows Neurotrace staining of a spinal cord slice. Scale bar = 50 μm. c and d Expression levels of c Rbfox3 and d Chat were unaltered after efnA5 knockdown in mice at 130 days of age. Data represents mean ± SEM ( N = 6–7), and different conditions were compared with a one-way ANOVA with Tukey’s multiple comparisons test: * P < 0.05; ** P < 0.01; *** P < 0.001 as compared with SOD1 WT mice. e Neuromuscular junctions (NMJs) innervation was scored in the gastrocnemius muscle. Data represents mean ± SEM ( N = 5–6) and it was analysed with a two-tailed t-test. Representative images are shown of denervated and innervated NMJs in the gastrocnemius muscle that were stained with neurofilament-L (NfL) and α-bungarotoxin (BTX). Scale bar = 50 μm

Article Snippet: For multiple reaction monitoring (MRM) analysis of efnA5 in CSF samples, 200 μL of CSF were spiked with TEAB buffer and a quantitative protein epitope signature tag (QPrEST, Atlas Antibodies AB, #QPrEST25042) of efnA5 as internal standard.

Techniques: Staining, Expressing, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

doi: 10.1186/s40478-019-0759-6

Figure Lengend Snippet: Reduction of efnA5 levels does not alter glial gene expression in symptomatic mice. Gene expression of several glial genes was quantified in the lumbar spinal cord of SOD1 WT , SOD1 G93A EfnA5 +/+ and SOD1 G93A EfnA5 +/− mice. a Aif , b Gfap , and c Vimentin levels were unaltered in SOD1 G93A EfnA5 +/− mice. Data represents mean ± SEM ( N = 6–7), and different conditions were compared with a one-way ANOVA with Tukey’s multiple comparisons test: * P < 0.05; ** P < 0.01; *** P < 0.001 as compared with SOD1 WT mice

Article Snippet: For multiple reaction monitoring (MRM) analysis of efnA5 in CSF samples, 200 μL of CSF were spiked with TEAB buffer and a quantitative protein epitope signature tag (QPrEST, Atlas Antibodies AB, #QPrEST25042) of efnA5 as internal standard.

Techniques: Expressing

Journal: Acta Neuropathologica Communications

Article Title: Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

doi: 10.1186/s40478-019-0759-6

Figure Lengend Snippet: Whole proteome is not altered in the lumbar spinal cord of SOD1 G93A EfnA5 +/− compared to SOD1 G93A EfnA5 +/+ mice at 130 days of age. Differences at a proteomic level were determined for a SOD1 G93A EfnA5 +/+ versus SOD1 WT , and for b SOD1 G93A EfnA5 +/− versus SOD1 G93A EfnA5 +/+ mice. Data is represented in Volcano plots and the black lines indicate the significance level based on an FDR of 5% and S0 = 0.1 to correct for multiple testing

Article Snippet: For multiple reaction monitoring (MRM) analysis of efnA5 in CSF samples, 200 μL of CSF were spiked with TEAB buffer and a quantitative protein epitope signature tag (QPrEST, Atlas Antibodies AB, #QPrEST25042) of efnA5 as internal standard.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

doi: 10.1186/s40478-019-0759-6

Figure Lengend Snippet: Axonal regeneration is not enhanced in EfnA5 knockout mice after sciatic nerve crush. Percentage of re-innervated neuromuscular junctions (NMJs) was determined in the a gastrocnemius and in the b tibialis anterior muscles in the ipsilateral and contralateral limbs of EfnA5 +/+ and EfnA5 −/− mice 11 days after the crush ( N = 5–5). Data represents mean ± SEM, and different conditions were compared with a two-tailed t-test. Representative images of ipsilateral and contralateral gastrocnemius and tibialis anterior muscles that were stained with neurofilament-L (NfL) and α-bungarotoxin (BTX) are shown. Scale bar = 50 μm

Article Snippet: For multiple reaction monitoring (MRM) analysis of efnA5 in CSF samples, 200 μL of CSF were spiked with TEAB buffer and a quantitative protein epitope signature tag (QPrEST, Atlas Antibodies AB, #QPrEST25042) of efnA5 as internal standard.

Techniques: Knock-Out, Two Tailed Test, Staining

Journal: Acta Neuropathologica Communications

Article Title: Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis

doi: 10.1186/s40478-019-0759-6

Figure Lengend Snippet: Disease progression is more severe in patients with lower CSF efnA5 protein levels. a Disease onset and b disease progression were determined in patients with CSF efnA5 levels above the median and in patients with CSF efnA5 levels below the median ( N = 32). Differences were compared between the two genotypes with the Log-rank test: ** p < 0.01

Article Snippet: For multiple reaction monitoring (MRM) analysis of efnA5 in CSF samples, 200 μL of CSF were spiked with TEAB buffer and a quantitative protein epitope signature tag (QPrEST, Atlas Antibodies AB, #QPrEST25042) of efnA5 as internal standard.

Techniques:

Journal: BMC Cancer

Article Title: Expression characteristic, immune signature, and prognosis value of EFNA family identified by multi-omics integrative analysis in pan-cancer

doi: 10.1186/s12885-022-09951-0

Figure Lengend Snippet: The clinical value of EFNA family in cancers. A EFNAs mRNA expression correlated with OS, DSS, DFI, and PFI from TCGA. B EFNAs mRNA expression correlated with OS and RFS in various cancers from GEO and other datasets. The prominently colored labels indicated p < 0.05. C Diagnostic ROC for EFNAs in LUAD and normal samples by TCGA combined with GTEx. D The overall survival curve of EFNA5 was analyzed integrated by the three LUAD data sets in GEO. E The nomogram for predicting 1 -, 3-and 5-year overall survival probability in LUAD patients. F Calibration of the nomogram for cox regression model and actual situation fitting analysis. G Univariate survival analysis of EFNA5 expression in subgroups with different characteristics

Article Snippet: The membrane was incubated at 4°C overnight with EFNA5 antibody (1:1000, Abmart, China) and β-tubulin antibody (Abways technology, China).

Techniques: Expressing, Diagnostic Assay

Journal: BMC Cancer

Article Title: Expression characteristic, immune signature, and prognosis value of EFNA family identified by multi-omics integrative analysis in pan-cancer

doi: 10.1186/s12885-022-09951-0

Figure Lengend Snippet: The relation between EFNA1/2/3/4/5 expression and clinical characteristics in LUAD in TCGA

Article Snippet: The membrane was incubated at 4°C overnight with EFNA5 antibody (1:1000, Abmart, China) and β-tubulin antibody (Abways technology, China).

Techniques: Expressing, Significance Assay

Journal: BMC Cancer

Article Title: Expression characteristic, immune signature, and prognosis value of EFNA family identified by multi-omics integrative analysis in pan-cancer

doi: 10.1186/s12885-022-09951-0

Figure Lengend Snippet: Univariate and multivariate analyses of overall survival in patients with LUAD in TCGA

Article Snippet: The membrane was incubated at 4°C overnight with EFNA5 antibody (1:1000, Abmart, China) and β-tubulin antibody (Abways technology, China).

Techniques:

Journal: BMC Cancer

Article Title: Expression characteristic, immune signature, and prognosis value of EFNA family identified by multi-omics integrative analysis in pan-cancer

doi: 10.1186/s12885-022-09951-0

Figure Lengend Snippet: Preliminary experimental verification of characteristics of EFNA5. A EFNA5 mRNA expression level in NSCLC cell lines from CCLE. B The expression level of EFNA5 in a normal lung bronchial epithelial cell line (HBE) and NSCLC cell lines was determined by Western Blot. C Immunocytochemistry for determining the subcellular location of EFNA5 in A431 and U251MG cell lines by HPA. EFNA5 localized to the membrane, cytosol, and vesicles (green). Microtubules are stained in red and the nucleus in blue (DAPI). D The mRNA relative expression level of EFNA5 on 13 LUAD and para-cancer paired tissues was determined by RT-qPCR. F Representative images of TMA stained with H&E and IHC for EFNA5. G EFNA5 expression levels in LUAD and peritumoral tissues determined in TMA. H EFNA5 staining score increased significantly with the characteristics of the primary tumor. I Overall survival analysis of 92 LUAD patients according to EFNA5 expression. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The membrane was incubated at 4°C overnight with EFNA5 antibody (1:1000, Abmart, China) and β-tubulin antibody (Abways technology, China).

Techniques: Expressing, Western Blot, Immunocytochemistry, Membrane, Staining, Quantitative RT-PCR