Journal: Scientific Reports

Article Title: Amyloid-β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization

doi: 10.1038/srep42548

Figure Lengend Snippet: ( a ) Pull-down assay of untagged Aβ 42 oligomers with purified NRX1βS4(−)-Fc proteins. Full gel blots for the cropped blots ( a ) are shown in the . ( b ) Saturable binding of biotin-Aβ 42 oligomers to COS-7 cells expressing HA-NRX1βS4(−). Data are presented as mean ± SEM. ( c ) Scatchard plot of binding data shown in ( b) . The Kd = 183.5 nM monomer equivalent. ( n = 30 cells for each plot).

Article Snippet: Purified soluble recombinant human NRX1βS4(−) ectodomain fused to human Fc (NRX1βS4(−)-Fc, 5268-NX-050, R&D systems) or human Fc (a negative control) generated from the pc4-sp-Fc vector were used for the pull-down assays.

Techniques: Pull Down Assay, Purification, Binding Assay, Expressing

Journal: Scientific Reports

Article Title: Amyloid-β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization

doi: 10.1038/srep42548

Figure Lengend Snippet: ( a,c ) Representative images of triple labeling for bound Fc proteins, bound Aβ 42 peptides and surface HA on COS-7 cells expressing the indicated extracellularly HA-tagged neurexin (NRX)1β construct. Recombinant neuroligin1-Fc (NLG1-Fc; 20 nM) binds to COS-7 cells expressing HA-NRX1βS4(+) or HA-NRX1βS4(−) but not to those expressing HA-NRX1βS4(−)D137A ( a ), and recombinant LRRTM2-Fc (20 nM) binds to COS-7 cells expressing HA-NRX1βS4(−) but not to those expressing HA-NRX1βS4(+) or HA-NRX1βS4(−)D137A ( c ). Treatment with Aβ 42 oligomers (AβOs, 500 nM, monomer equivalent) does not seem to affect the binding in any of these cases. Scale bars represent 30 μm. ( b,d ) Quantification of recombinant NLG1-Fc ( b ) or LRRTM2-Fc ( d ) bound to COS-7 cells expressing the indicated HA-NRX1β constructs treated with vehicle control or 500 nM Aβ 42 oligomers. n = 30 cells for each condition from three independent experiments, one-way ANOVA, P < 0.0001. n.s., not significant by Bonferroni multiple comparisons test. Data are presented as mean ± SEM.

Article Snippet: Purified soluble recombinant human NRX1βS4(−) ectodomain fused to human Fc (NRX1βS4(−)-Fc, 5268-NX-050, R&D systems) or human Fc (a negative control) generated from the pc4-sp-Fc vector were used for the pull-down assays.

Techniques: Labeling, Expressing, Construct, Recombinant, Binding Assay

Journal: Scientific Reports

Article Title: Amyloid-β Oligomers Interact with Neurexin and Diminish Neurexin-mediated Excitatory Presynaptic Organization

doi: 10.1038/srep42548

Figure Lengend Snippet: ( a–c ) Representative time-lapse images of axons expressing extracellularly super-ecliptic pHluorin (SEP)-tagged NRX1βS4(+) ( a ), SEP-NRX1βS4(−) ( b ) and SEP-NRX1βS4(−) lacking the histidine-rich domain (SEP-NRX1βS4(−)∆HRD) ( c ) treated with Aβ 42 oligomers (500 nM, monomer equivalent) at t = 0 min. Scale bars represent 5 μm. ( d ) Quantification of SEP intensity signals at 5 min before and 10, 30 and 60 min after Aβ 42 oligomer treatment. n = 29 for SEP-NRX1βS4(+), n = 26 for SEP-NRX1βS4(−), and n = 33 for SEP-NRX1βS4(−)ΔHRD puncta from 9 cells for each condition from three independent experiments, two-way repeated measures ANOVA, F (3, 255) = 42.94, P < 0.0001 for time and F (2, 85) = 18.95, P < 0.0001 for construct. ** P < 0.001, and *** P < 0.0001 compared with SEP signal at 5 min before the treatment, and *** P < 0.0001 compared with SEP-NRX1βS4(−)ΔHRD by Bonferroni multiple comparisons tests. n.s., not significant. Data are presented as mean ± SEM.

Article Snippet: Purified soluble recombinant human NRX1βS4(−) ectodomain fused to human Fc (NRX1βS4(−)-Fc, 5268-NX-050, R&D systems) or human Fc (a negative control) generated from the pc4-sp-Fc vector were used for the pull-down assays.

Techniques: Expressing, Construct