wue4 capture antibody cat# nb110-60531  (Novus Biologicals)

Journal: Nature Communications

Article Title: APOE ε2 is associated with increased tau pathology in primary tauopathy

doi: 10.1038/s41467-018-06783-0

Figure Lengend Snippet: Behavioral abnormalities and compromised synaptic integrity in Tau P301L -apoE2 mice. Behavioral performance was assessed in control mice (Ctrl, AAV-GFP-injected, n = 12 mice per group, mixed gender) and Tau P301L -apoE2, -apoE3, and -apoE4 mice ( n = 13 mice for Tau P301L -apoE2 group, n = 20 mice for Tau P301L -apoE3 group, n = 22 mice for Tau P301L -apoE4 group, mixed gender) at 6 months of age. a Open-field analysis was assessed and the ratios of time spent in the center quadrant to total distance traveled in the open-field apparatus are shown. b Exploratory behavior was evaluated in the elevated plus maze and the ratios of the time spent in open arms to close arms are shown. c , d Fear conditioning test was utilized to examine the associative memory. The percentage of the time with freezing behavior in response to stimulus during context ( c ) and cued ( d ) tests is shown. Data represent mean ± SEM. e – g The amount of GluR2 and PSD95 in the cortical RIPA lysate was examined by western blotting for control mice and Tau P301L -apoE mice ( n = 6 mice per group, mixed gender) at 6 months of age. Results were normalized to β-actin levels. Data are expressed as mean ± SEM. Mann-Whitney tests followed by Bonferroni correction for multiple comparisons were used. * P < 0.0167; N.S. not significant

Article Snippet: For apoE ELISA, WUE4 capture antibody (Cat# NB110-60531, Novus, 1:1000) and biotin-conjugated mouse monoclonal detector antibody (Cat# K74180B, Meridian Life Science, 1:10 000) were used as described .

Techniques: Control, Injection, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: APOE ε2 is associated with increased tau pathology in primary tauopathy

doi: 10.1038/s41467-018-06783-0

Figure Lengend Snippet: Increased insolubility of tau and apoE in Tau P301L -apoE2 mice. The cortical brain tissues from control mice (Ctrl, AAV-GFP-injected) and Tau P301L -apoE2, -apoE3, and -apoE4 mice at 6 months of age were sequentially extracted by RAB, RIPA, and FA buffer. Soluble tau (HT7 detection) and apoE in RAB ( a , b ) and RIPA fractions ( a , c ) were examined by western blotting ( n = 6 mice per group, mixed gender). Results were normalized to β-actin levels. Insoluble tau and apoE in FA fraction was detected by ELISA ( d n = 6 mice per group, mixed gender). Data are expressed as mean ± SEM relative to apoE2-TR mice. Mann-Whitney tests followed by Bonferroni correction for multiple comparisons were used. * P < 0.0167; N.S. not significant

Article Snippet: For apoE ELISA, WUE4 capture antibody (Cat# NB110-60531, Novus, 1:1000) and biotin-conjugated mouse monoclonal detector antibody (Cat# K74180B, Meridian Life Science, 1:10 000) were used as described .

Techniques: Control, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: APOE ε2 is associated with increased tau pathology in primary tauopathy

doi: 10.1038/s41467-018-06783-0

Figure Lengend Snippet: Disulfide bond-mediated complex formation between tau and apoE in vitro. The in vitro interaction between tau and apoE proteins was evaluated by solution binding assay, followed by western blotting (four independent experiments). Recombinant human apoE protein was incubated with tau protein for 1 h at 37 °C in 20 µl of phosphate-buffered saline. The reaction was quenched by addition of 20 µl of sample buffer without (lane 1–7) or with (lane 8–10) reducing agent 2-mercaptoethanol (2-ME). a The presence of tau/apoE complexes (bands A and B) were determined by immunoblotting with antibodies to tau and apoE, respectively. The amounts of tau ( b ) and apoE ( c ) in the tau/apoE complexes were quantified. Bands A and B were tau/apoE complexes. Band C was a nonspecific band from tau immunoblot. Bands D and F were monomeric tau and apoE, respectively. Band E was estimated to be apoE dimer. Data are expressed as mean ± SEM relative to apoE2 condition. One-way ANOVA with Tukey post hoc tests were used. * P < 0.05; N.S. not significant

Article Snippet: For apoE ELISA, WUE4 capture antibody (Cat# NB110-60531, Novus, 1:1000) and biotin-conjugated mouse monoclonal detector antibody (Cat# K74180B, Meridian Life Science, 1:10 000) were used as described .

Techniques: In Vitro, Solution Binding Assay, Western Blot, Recombinant, Incubation, Saline

Journal: Nature Communications

Article Title: APOE ε2 is associated with increased tau pathology in primary tauopathy

doi: 10.1038/s41467-018-06783-0

Figure Lengend Snippet: Associations of APOE genotype with tau lesions of PSP

Article Snippet: For apoE ELISA, WUE4 capture antibody (Cat# NB110-60531, Novus, 1:1000) and biotin-conjugated mouse monoclonal detector antibody (Cat# K74180B, Meridian Life Science, 1:10 000) were used as described .

Techniques:

Journal: Nature Communications

Article Title: APOE ε2 is associated with increased tau pathology in primary tauopathy

doi: 10.1038/s41467-018-06783-0

Figure Lengend Snippet: Associations of APOE genotype with risk of PSP and CBD

Article Snippet: For apoE ELISA, WUE4 capture antibody (Cat# NB110-60531, Novus, 1:1000) and biotin-conjugated mouse monoclonal detector antibody (Cat# K74180B, Meridian Life Science, 1:10 000) were used as described .

Techniques:

Journal: Neuron

Article Title: Apolipoprotein E4 impairs neuronal insulin signaling by trapping insulin receptor in the endosomes

doi: 10.1016/j.neuron.2017.09.003

Figure Lengend Snippet: (A and B) The amount of hippocampal p-Akt (Ser473), total Akt and β-actin in old (22 months) apoE-TR mice (A) or HFD-treated middle-age (12 months) apoE-TR mice (B) was examined by Western blotting after insulin treatment via reverse microdialysis (n=4 mice/genotype, male). Results were normalized to β-actin levels. Both contralateral (without insulin) and ipsilateral (with insulin) hippocampal homogenates from the same animal were analyzed on the same blot and the change in p-Akt/total Akt ratio in response to insulin treatment was calculated. Data were normalized to apoE3-TR mice for comparison. (C) Apoe−/− primary neurons were treated overnight with 50 nM recombinant apoE3 or apoE4 followed by insulin treatment (100 nM) for 30 minutes. The amount of p-Akt (Ser473), total Akt and β-actin was detected by Western blotting (three independent experiments). The change in p-Akt/total Akt ratio after insulin treatment was calculated. Data were normalized to apoE3 treatment for comparison. Values are expressed as mean ± SEM. Two-tailed student’s t test was used for statistical analysis. *p < 0.05. Molecular mass markers in kilodaltons are shown.

Article Snippet: For apoE ELISA, WUE4 capture antibody (Novus) and biotin-conjugated mouse monoclonal K74180B detector antibody (Meridian Life Science) were used as described ( Fu et al., 2016 ).

Techniques: Western Blot, Comparison, Recombinant, Two Tailed Test

Journal: Neuron

Article Title: Apolipoprotein E4 impairs neuronal insulin signaling by trapping insulin receptor in the endosomes

doi: 10.1016/j.neuron.2017.09.003

Figure Lengend Snippet: Mitochondrial respiration and glycolysis were measured by Seahorse XFe96 analyzer in Apoe−/− neurons treated overnight with vehicle or 50 nM recombinant apoE3 or apoE4 followed by insulin (100 nM) treatment for 30 minutes. (A–C) Oxygen consumption rate (OCR) was assessed over time after sequential injections of 2 μM oligomycin, 1 μM FCCP, and 5 μM rotenone/antimycin A. The basal respiration and the maximal respiration were calculated to compare the effect of insulin treatment. (D–F) The extracellular acidification rate (ECAR) was assessed over time after sequential injections of glucose (10 mM), oligomycin (2 μM) and 2-DG (50 mM). The glycolysis rate and glycolytic capacity were calculated to compare the effect of insulin treatment. Each value was derived from 10 to 12 repeats in two independent experiments and normalized to the third data point measurement of baseline from non-insulin treatment group for comparisons. Data are expressed as mean ± SEM. Two-tailed student’s t test was used for statistical analysis. ****p < 0.0001; N.S., not significant. Oligo, Oligomycin; FCCP, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; RA, rotenone/antimycin A.

Article Snippet: For apoE ELISA, WUE4 capture antibody (Novus) and biotin-conjugated mouse monoclonal K74180B detector antibody (Meridian Life Science) were used as described ( Fu et al., 2016 ).

Techniques: Recombinant, Derivative Assay, Two Tailed Test

Journal: Neuron

Article Title: Apolipoprotein E4 impairs neuronal insulin signaling by trapping insulin receptor in the endosomes

doi: 10.1016/j.neuron.2017.09.003

Figure Lengend Snippet: (A and B) The interaction between IR and recombinant apoE (A) or astrocyte-secreted lipidated apoE particles (B) were evaluated by solid-phase binding assay (three independent experiments). The Kd values of the binding curves were calculated using the one-site specific binding equation. (C) The interaction between IR and recombinant apoE was evaluated by solution binding assay, followed by immunoprecipitation (IP) of apoE and immunoblotting (IB) of IR. The ratio of IR to apoE was calculated. The values were normalized to apoE3 for comparison. (D–E) The domain interaction between apoE and IR was determined by solid-phase binding assay (three independent experiments) using C-terminal truncated apoE3 or apoE4 containing the aminol acid (aa) 1-186 compared with the full-length (FL) of apoE. The values were normalized to the binding of apoE FL for comparison. (F) A competitive inhibition assay of insulin (8 nM), IR (80 nM) and apoE (4, 8, or 16 μg/ml) was analyzed using solid-phase binding assay (three independent experiments). Results were normalized to the maximal binding of insulin and IR in the absence of apoE. (G–K) Primary Apoe−/− neurons were treated overnight with recombinant apoE3 or apoE4 (100 nM) in the presence or absence of insulin (100 nM) stimulation for 30 minutes, followed by cell surface biotinylation assay and Western blot analysis (three independent experiments). The cell surface IR and total IR were shown and the ratio of cell surface IR/total IR was calculated (G, H). The values were normalized to the vehicle-treated group (V) for comparison. The change in cell surface IR/total IR after insulin treatment was evaluated (I). The amount of p-Akt (Ser473), total Akt and β-actin was detected by Western blotting (G, J, K). Ratio of p-Akt to total Akt (J) and insulin-induced p-Akt/Akt change (K) were calculated. The values were normalized to vehicle without insulin treatment group for comparison. All data represent mean ± SEM. Two-tailed student’s t test (A–C, F, I, K) and one-way ANOVA with Tukey’s multiple comparisons test (D, E, H, J) were used in for statistical analysis. *p < 0.05; **p < 0.01; N.S., not significant. Molecular mass markers in kilodaltons are shown.

Article Snippet: For apoE ELISA, WUE4 capture antibody (Novus) and biotin-conjugated mouse monoclonal K74180B detector antibody (Meridian Life Science) were used as described ( Fu et al., 2016 ).

Techniques: Recombinant, Binding Assay, Solution Binding Assay, Immunoprecipitation, Western Blot, Comparison, Inhibition, Cell Surface Biotinylation Assay, Two Tailed Test

Journal: Neuron

Article Title: Apolipoprotein E4 impairs neuronal insulin signaling by trapping insulin receptor in the endosomes

doi: 10.1016/j.neuron.2017.09.003

Figure Lengend Snippet: RIPA and guanidine (GND)-extracted brain cortical lysates of apoE3-TR and apoE4-TR mice (n=4–6 mice/genotype, mixed gender) at 3 and 22 months of age were prepared and the amount of apoE and IR was examined by Western blotting (A–C) and ELISA (D–I). Results were normalized to β-actin levels in Western blot analysis. The ratios of insoluble (GND fraction) to soluble (RIPA fraction) apoE (F) and IR (I) were calculated. Data are expressed as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis. **p < 0.01; ***p < 0.001, N.S., not significant. Molecular mass markers in kilodaltons are shown.

Article Snippet: For apoE ELISA, WUE4 capture antibody (Novus) and biotin-conjugated mouse monoclonal K74180B detector antibody (Meridian Life Science) were used as described ( Fu et al., 2016 ).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay