notch3 ecd 1e4  (Merck & Co)

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: The distribution of NOTCH3 cys variants in CADASIL cohorts and in population databases and the NVFOR per EGFr domain. ( A ) Distribution of NOTCH3 cys variants in CADASIL cohorts ( top ) and population databases ( bottom ) along the 34 EGFr domains. ( B ) Interval plot showing the NVFOR of each EGFr domain with error bars indicating 95% confidence intervals. Each EGFr domain is classified in an EGFr risk category, based on their respective NVFOR: HR-EGFr domains have NVFOR >5, MR-EGFr domains have an NVFOR between 0.20 and 5 and LR-EGFr domains have an NVFOR <0.20. If an EGFr domain included less than 10 NOTCH3 cys variant counts, then it was classified as an UR-EGFr domain. The dotted lines indicate NVFOR 0.2 and 5.0, which are the cut-off points used for the EGFr risk categories.

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Variant Assay

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: Distribution of high-risk, medium-risk and low-risk EGFr domains along the NOTCH3 protein and in CADASIL and population genotype–phenotype data sets. ( A ) Schematic representation of the transmembrane NOTCH3 protein. The NOTCH3 gene contains 33 exons, of which exons 2–24 encode the 34 EGFr domains located in the extracellular domain. Each EGFr domain is classified in an EGFr risk category based on their respective NVFOR. ( B ) Proportional bar charts showing the proportion of individuals with a NOTCH3 cys variant for each EGFr risk category in the CADASIL and population genotype–phenotype data sets. Most CADASIL patients harbour an HR-EGFr variant (66.1%), whereas the majority of individuals in the population genotype–phenotype data set harbour an LR-EGFr variant (85.7%).

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Variant Assay

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: Characteristics of individuals with a NOTCH3 cys variant in the CADASIL and population data sets

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Variant Assay

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: CADASIL patients with a NOTCH3 cys variant located in the newly identified HR-EGFr domains 8, 11 and 26 are clinically and radiologically as severe as patients with HR-EGFr 1–6 variants . Two scatterplots and three interval plots showing nWMHv ( A ), PSMD ( B ), nLV ( C ), risk of stroke ( D ) and disability ( E ) stratified per EGFr risk category in the CADASIL genotype–phenotype data set after correction for cardiovascular risk factors and sex. CADASIL patients with NOTCH3 cys variants located in the newly identified HR-EGFr domains 8, 11 and 26 had a significantly higher nWMHv ( P = 1.8 × 10 −8 ), PSMD ( P = 2.6 × 10 −8 ), nLV ( P = 0.006), risk of stroke ( P = 0.002) and disability ( P = 0.041) than CADASIL patients with MR-EGFr variants. There were no significant differences between patients with HR-EGFr 1–6 variants and those with variants in HR-EGFR domains 8, 11 and 26 in nWMHv ( P = 0.27), PSMD ( P = 0.087), nLV ( P = 0.87), disability ( P = 0.26) and risk of stroke ( P = 0.75). Error bars indicate 95% confidence interval of the group means. Statistical significance after correction for multiple testing: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Variant Assay

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: NOTCH3 cys variants in EGFr domains 1–5, 14 and 26 are most frequent in the CADASIL genotype–phenotype data set: associations with disease severity . Five interval plots showing the estimated marginal means, odds ratios or hazard ratios of SVD imaging markers, risk of stroke and disability for the EGFr domains most frequently harbouring NOTCH3 cys variants in the CADASIL genotype–phenotype data set, after correction for cardiovascular risk factors and sex. Only EGFr domains which had high NOTCH3 cys variant counts (≥10 individuals for SVD imaging markers and ≥20 individuals for clinical outcomes) were included in the analyses. Error bars indicate 95% confidence intervals of the group means. ( A – D ) Of all frequently mutated HR-EGFr domains, EGFr domains 2 and 3 were associated with the highest burden of nWMHv, PSMD, nLV and risk of stroke. There was an evident decrease in the SVD imaging marker loads and risk of stroke in EGFr domain 3 through EGFr domain 5. EGFr domain 26 was associated with as high a burden of SVD imaging markers and risk of stroke as the other HR-EGFr domains. EGFr domain 14, which was the only MR-EGFr domain with a high frequency of NOTCH3 cys variants in CADASIL patients, was associated with the lowest burden of SVD imaging markers and risk of stroke. ( E ) There were no significant differences in disability. Further details including statistically significant differences in the burden of SVD imaging markers and risk of stroke between EGFr domains are shown in .

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Imaging, Variant Assay, Marker

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: The risk of stroke between the NOTCH3 cys EGFr risk categories in the CADASIL and population genotype–phenotype data set. A survival curve ( A ) and interval plot ( B ), both corrected for cardiovascular risk factors and sex, showing that MR-EGFr and HR-EGFr patients in the CADASIL genotype–phenotype data set had a significant earlier onset of stroke than individuals from the same EGFr risk category in the population genotype–phenotype data set [MR-EGFr: median 72 versus >78 years ( P = 8.1 × 10 −6 ); HR-EGFr: median 57 versus 74 years ( P = 3.5 × 10 −5 )]. The risk of stroke of HR-EGFr individuals in the population data set and MR-EGFr patients from the CADASIL data set was similar ( P = 0.54).

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques:

Journal: Brain

Article Title: Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction

doi: 10.1093/brain/awac486

Figure Lengend Snippet: Differences in NOTCH3 aggregation and signalling between EGFr risk categories. ( A – C ) NOTCH3 aggregation load, expressed as immunohistochemical NOTCH3 score, was determined in skin tissue samples of LR-EGFr ( n = 3), MR-EGFr ( n = 6) and HR-EGFr ( n = 12) patients, of which six had an HR-EGFr 1–6 NOTCH3 cys variant and six had a NOTCH3 cys variant located in the newly identified HR-EGFr domain 26. ( A ) As there were only tissue samples available of three LR-EGFr patients, these patients were excluded from the group comparisons. HR-EGFr 1–6 CADASIL patients had a significantly higher NOTCH3 score than MR-EGFr patients: mean NOTCH3 score 43.0 (95% CI: 30.3–55.6) versus 23.2 (95% CI: 10.5–35.8) ( P = 0.033). HR-EGFr 26 patients had a mean NOTCH3 score of 32.8 (95% CI: 20.1–45.5), which did not significantly differ from that of HR-EGFr 1–6 ( P = 0.24) and MR-EGFr ( P = 0.27) patients. The three LR-EGFr patients had a mean NOTCH3 score of 15.4 (95% CI: 0–32.6). ( B ) NVFOR was significantly associated with a NOTCH3 score ( P = 0.003), also after correction for age ( P = 0.006). ( C ) Representative images of NOTCH3-immunohistochemistry of skin biopsies of HR-EGFr 1–6, HR-EGFr 26, MR-EGFr and LR-EGFr patients, showing increasing granular NOTCH3 staining from low- to high-risk EGFr domains. Bar represents 20 µm. ( D ) NOTCH3 signalling activity was assessed in NIH 3T3 cells using a CBF1-responsive luciferase assay with and without Jagged1 stimulation. NOTCH3 signalling was expressed as fold increase of luciferase activity upon Jagged1 stimulation (= ‘luciferase activity with Jagged1 stimulation’/‘luciferase activity without Jagged1 stimulation’). Each dot represents an individual experiment. Signalling activity did not differ significantly between NOTCH3 cys variants located in the other HR-EGFr domains and the MR-EGFr and LR-EGFr domains (all P > 0.58). However, NOTCH3 cys variants in HR-EGFr and MR-EGFr showed a significantly lower NOTCH3 signalling activity compared to wild-type ( P = 0.008 and P = 0.02). As expected, NOTCH3 cys variants located in the HR-EGFr domain 11 had strongly impaired ligand-induced signalling activity, comparable to a deletion of the ligand binding domain ( P = 0.53). There was no significant difference in NOTCH3 signalling activity between HR-EGFr domain 26 and other HR-EGFr domains, MR-EGFr domains and LR-EGFr domains (all P > 0.47). ( E ) NVFOR was not associated with NOTCH3 signalling activity ( P = 0.88). Error bars indicate 95% confidence intervals of the group means. Statistical significance after correction for multiple testing: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Skin tissue samples were processed and stained with a monoclonal antibody targeting NOTCH3 ECD (clone 1E4, Millipore), full-focus microscopy images were taken and NOTCH3 ECD -positive area was quantified in all visible blood vessel walls (median 30, range 21–37) using ImageJ, as previously described.

Techniques: Immunohistochemical staining, Variant Assay, Immunohistochemistry, Staining, Activity Assay, Luciferase, Ligand Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF 1–5 ‐immunized mice at 7 months of age and TgN3R182C150 at 18 months of age. Representative images show brain arteries of TgN3R182C150 (7 and 18 months), sham and NOTCH3 EGF 1–5 ‐immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α‐SMA antibody (green). Scale bar = 20 μm. Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF 1–5 ‐immunized ( n = 10), sham ( n = 8), and non‐vaccinated ( n = 6) TgN3R182C150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months ( n = 3) of age versus NOTCH3 EGF 1–5 ‐immunized ( n = 10), sham ( n = 8), and non‐vaccinated ( n = 6) TgN3R182C150 mice at 7 months of age. Statistical significance was assessed using a Brown‐Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons (NOTCH3 ECD deposits (% of vessel area): 7 m.o. vs. Sham, ns P = 0.9424; 7 m.o. vs. Vaccinated, ns P = 0.9809; 7 m.o. vs. 18 m.o. ** P = 0.0046; Sham vs. Vaccinated, ns P = 0.9999; Sham vs. 18 m.o. ** P = 0.0032; Vaccinated vs. 18 m.o. ** P = 0.0032. NOTCH3 ECD deposits (number/1,000 μm 2 ): 7 m.o. vs. Sham, ns P = 0.7877; 7 m.o. vs. Vaccinated, ns P = 0.8358; 7 m.o. vs. 18 m.o. ** P = 0.0069; Sham vs. Vaccinated, ns P = 0.9995; Sham vs. 18 m.o. ** P = 0.0044; Vaccinated vs. 18 m.o. * P = 0.0104). NOTCH3 ECD deposits size: 7 m.o. vs. Sham, ns P = 0.1169; 7 m.o. vs. Vaccinated, ns P > 0.9999; 7 m.o. vs. 18 m.o. *** P = 0.0009; Sham vs. Vaccinated, ns P = 0.0577; Sham vs. 18 m.o. ** P = 0.007; Vaccinated vs. 18 m.o. **** P < 0.0001, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: To assess the NOTCH3 ECD deposits, the tissues were incubated overnight at 4°C with mouse monoclonal anti‐human NOTCH3 ECD primary antibody (1:100, clone 1E4, Millipore) followed by detection with Alexa 594 conjugated anti‐mouse secondary antibody (1:1,000, Life Technologies).

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images of TgN3R182C150, sham‐ and NOTCH3 EGF 1–5 ‐immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF 1–5 ‐immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti‐perlecan antibody (green). Scale bar = 20 μm. Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3‐ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non‐vaccinated 3‐month‐old TgN3R182C150 ( n = 3) and 7‐month‐old TgN3R182C150 ( n = 6) mice and 18‐month‐old TgN3R182C150 ( n = 3). Quantification of NOTCH3‐ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3‐ECD stained area and average size per vessel revealed a significant decrease in NOTCH3‐ECD deposition in brain arteries and capillaries between NOTCH3 EGF 1–5 ‐immunized ( n = 11), sham ( n = 8), and non‐vaccinated TgN3R182C150 ( n = 6) mice. Statistical significance was assessed using a Brown‐Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons (NOTCH3 ECD deposits (% of vessel area): 3 m.o. vs. 18 m.o. ** P = 0.0029; 3 m.o. vs. 7 m.o. *** P = 0.0004; 7 m.o. vs. Sham, ns P = 0.7326; 7 m.o. vs. Vaccinated, ** P = 0.003; Sham vs. Vaccinated, **** P < 0.0001. NOTCH3 ECD deposits (number/1,000 μm 2 ): 3 m.o. vs. 18 m.o. ** P = 0.0038; 3 m.o. vs. 7 m.o. *** P = 0.0002; 7 m.o. vs. Sham, ns P = 0.9913; 7 m.o. vs. Vaccinated, * P = 0.021; Sham vs. Vaccinated, *** P = 0.001. NOTCH3 ECD deposits size: 3 m.o. vs. 18 m.o. * P = 0.0215; 3 m.o. vs. 7 m.o. ** P = 0.0029; 7 m.o. vs. Sham, ns P = 0.3622; 7 m.o. vs. Vaccinated, * P = 0.0428; Sham vs. Vaccinated, ** P = 0.0038, ns = non‐significant). Dotted lines indicate quartiles and dashed thicker lines are the median. Source data are available online for this figure.

Article Snippet: To assess the NOTCH3 ECD deposits, the tissues were incubated overnight at 4°C with mouse monoclonal anti‐human NOTCH3 ECD primary antibody (1:100, clone 1E4, Millipore) followed by detection with Alexa 594 conjugated anti‐mouse secondary antibody (1:1,000, Life Technologies).

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Active immunotherapy reduces NOTCH3 deposition in brain capillaries in a CADASIL mouse model

doi: 10.15252/emmm.202216556

Figure Lengend Snippet: Representative images show microglia stained with anti‐CD68 antibody (red) and Iba1 antibody (green). Scale bar = 20 μm. Quantification of CD68‐stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF 1–5 ‐immunized ( n = 6), sham ( n = 4), and non‐vaccinated TgN3R182C150 ( n = 5) mice. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Tukey's multiple comparisons test (% microglia with CD68 staining: 7 m.o. vs. Sham ns P = 0.7393; Sham vs. Vaccinated ** P = 0.0087. CD68 staining (% of microglia area): 7 m.o. vs. Sham ns P = 0.9131; Sham vs. Vaccinated * P = 0.0157, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Representative images of TgN3R182C150, sham‐, and NOTCH3 EGF 1–5 ‐immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). Scale bar = 20 μm. Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm 2 ) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF 1–5 ‐immunized ( n = 11), sham ( n = 8), and non‐vaccinated TgN3R182C150 ( n = 6) mice at 7 months of age. Statistical significance was assessed using an ordinary one‐way ANOVA followed by Tukey's multiple comparisons test (% microglia with NOTCH3 ECD deposits: 7 m.o. vs. Sham ns P = 0.7997; Sham vs. Vaccinated ns P = 0.0526. NOTCH3 ECD deposits (% of microglia area): 7 m.o. vs. Sham ns P = 0.5362; Sham vs. Vaccinated ns P = 0.8059. NOTCH3 ECD deposits (number/1,000 μm 2 ): 7 m.o. vs. Sham ns P = 0.8392; Sham vs. Vaccinated ns P = 0.5777. NOTCH3 ECD deposits size: 7 m.o. vs. Sham ns P = 0.6664; Sham vs. Vaccinated ns P = 0.8787, ns = non‐significant). Error bars indicate standard error of the mean (SEM). Source data are available online for this figure.

Article Snippet: To assess the NOTCH3 ECD deposits, the tissues were incubated overnight at 4°C with mouse monoclonal anti‐human NOTCH3 ECD primary antibody (1:100, clone 1E4, Millipore) followed by detection with Alexa 594 conjugated anti‐mouse secondary antibody (1:1,000, Life Technologies).

Techniques: Staining

Journal: Human Molecular Genetics

Article Title: Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients

doi: 10.1093/hmg/ddz285

Figure Lengend Snippet: Pedigree and brain imaging of individuals with the NOTCH3 c.1492G>T variant. ( A ) Pedigree showing the family members with the NOTCH3 c.1492G>T, p.G498C variant. ( B – E ) FLAIR sequences are shown, unless otherwise stated. (B) MRI of the index patient (#1) shows confluent symmetrical periventricular and subcortical WMH. WMH are also present in the anterior temporal lobes. No lacunes were seen. Microbleeds are present in the thalamus on susceptibility-weighted imaging (SWI) (B 2 ). (C) MRI scan of the twin sister (#2) showing confluent WMH almost identical to those seen in the index, and a parietal subcortical microbleed on the SWI scan, but no lacunes (C 2 ). (D) Brain MRI in the brother (#4) was performed at the age of 63 years, before his large vessel stroke, showing focal and beginning confluent WMH in the semioval centre with some involvement of external capsules, but without lacunes. (D 2 ) A CT scan made after the stroke reveals a large cortico-subcortical infarct in the territory of the left medial cerebral artery (arrowheads). (E) MRI of the nephew (#6) showing minimal focal WMH in the frontal deep white matter. No brain MRI was available for one brother (#5). ( F ) Violin plot showing age-adjusted standardized WMH scores of the index (#1), her twin sister (#2) and the nephew (#6) compared to a cohort of CADASIL patients. ( G ) Violin plot showing age-adjusted standardized lacune count scores in the family members #1, #2, #4 and #6. None of the family members had lacunes. The differences the standardized score for lacunes between family members are due to the differences in age (see ).

Article Snippet: In short, cells were either fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 or only fixed without permeabilization using 4% paraformaldehyde and stained with a mouse anti-NOTCH3 ECD antibody (clone 1E4, Millipore, dilution 1:10 000).

Techniques: Imaging, Variant Assay, Capsules, Computed Tomography

Journal: Human Molecular Genetics

Article Title: Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients

doi: 10.1093/hmg/ddz285

Figure Lengend Snippet: Exon 9 skipping and predicted effect on NOTCH3 protein of NOTCH3 c.1492G>T variant. ( A ) The NOTCH3 gene contains 33 exons, of which exon 2–24 encode the 34 EGFr domains in the ectodomain of the NOTCH3 protein (NOTCH3 ECD ). One exon can encode one or more, complete or partial EGFr domains. ( B ) The NOTCH3 c.1492G>T variant is located adjacent to the splice donor site of exon 9. Upon canonical splicing of the NOTCH3 transcript, the c.1492G>T variant results in an additional cysteine residue in EGFr domain 12. However, because the c.1492G>T variant is located adjacent to the splice donor site, it is predicted to result in exon 9 skipping. Exon 9 encodes part of EGFr 11 and part of EGFr 12; the remaining parts of EGFr 11 and EGFr 12 encoded by exon 8 and exon 10, respectively, are predicted to form an EGFr 11–12 fusion domain, with the correct number of and spacing of cysteine residues. Homology-based models show the predicted structure of EGFr domains. ( C ) RT-PCR and Sanger sequencing analysis showing exon 9 skipping in fibroblast RNA from individuals with the NOTCH3 c.1492G>T variant. The full-length band predominantly contained the wild-type transcript, but also some mutant transcripts, as indicated by the relatively low mutant T-peak in the sequence. No exon 9 skipping was seen in controls. RT-PCR and Sanger sequencing were performed in two independent experiments. NOTCH3 ECD , NOTCH3 extracellular domain. EGFr, Epidermal growth factor-like repeat.

Article Snippet: In short, cells were either fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 or only fixed without permeabilization using 4% paraformaldehyde and stained with a mouse anti-NOTCH3 ECD antibody (clone 1E4, Millipore, dilution 1:10 000).

Techniques: Variant Assay, Residue, Reverse Transcription Polymerase Chain Reaction, Sequencing, Mutagenesis

Journal: Human Molecular Genetics

Article Title: Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients

doi: 10.1093/hmg/ddz285

Figure Lengend Snippet: Individuals with the NOTCH3 c.1492G>T variant show much less granular NOTCH3 ECD staining in skin vasculature than CADASIL patients. ( A ) Representative images of NOTCH3 ECD staining in blood vessels of skin punch biopsies: CADASIL patients, individuals with the NOTCH3 c.1492G>T variant and controls. All family members with the NOTCH3 c.1492G>T variant had minimal granular NOTCH3 ECD staining, which was much less compared to NOTCH3 ECD staining typically seen in CADASIL patients. No NOTCH3 ECD staining was seen in the brother without the NOTCH3 variant (#3) or in controls. ( B ) Quantification of the area of NOTCH3 ECD immunostaining within the vessel wall showed that individuals with the NOTCH3 c.1492G>T variant have significantly less NOTCH3 ECD staining compared to CADASIL patients (1.81% ± 1.48 versus 31.2% ± 2.75 NOTCH3 ECD positive area within vessel wall boundaries, ANOVA, P < 0.001). Bar represents 10 μm. Mean ± standard deviation.

Article Snippet: In short, cells were either fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 or only fixed without permeabilization using 4% paraformaldehyde and stained with a mouse anti-NOTCH3 ECD antibody (clone 1E4, Millipore, dilution 1:10 000).

Techniques: Variant Assay, Staining, Immunostaining, Standard Deviation

Journal: Human Molecular Genetics

Article Title: Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients

doi: 10.1093/hmg/ddz285

Figure Lengend Snippet: NOTCH3 Δexon9 protein has normal cellular localisation but has reduced signalling properties in vitro . ( A ) Immunocytochemistry of fibroblasts transfected with NOTCH3 cDNA constructs shows that the NOTCH3 Δexon9 protein is translated and expressed on the cell surface similar to NOTCH3 WT , as indicated by NOTCH3 ECD staining of the cell membrane in unpermeabilized cells. ( B ) NOTCH3 signalling in a CBF1 reporter assay with and without JAGGED1 stimulation. JAGGED1 significantly activated NOTCH3 signalling in NOTCH3 WT , NOTCH3 Y465C and NOTCH3 G498C , but not in NOTCH3 Δexon9 and NOTCH3 ΔLBD (independent samples t -test; see for details). RLU, relative luciferase units; N3 WT , NOTCH3 wild-type protein; NOTCH3 Δexon9 , NOTCH3 exon 9 skip protein; scale bar represents 50 μm. Mean ± standard deviation of seven independent experiments. ** P < 0.01, *** P < 0.001.

Article Snippet: In short, cells were either fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 or only fixed without permeabilization using 4% paraformaldehyde and stained with a mouse anti-NOTCH3 ECD antibody (clone 1E4, Millipore, dilution 1:10 000).

Techniques: In Vitro, Immunocytochemistry, Transfection, Construct, Staining, Membrane, Reporter Assay, Luciferase, Standard Deviation

Journal: Human Molecular Genetics

Article Title: Naturally occurring NOTCH3 exon skipping attenuates NOTCH3 protein aggregation and disease severity in CADASIL patients

doi: 10.1093/hmg/ddz285

Figure Lengend Snippet: Targeted NOTCH3 exon exclusion using ASOs and CRISPR/Cas9 in vitro . ( A ) RT-PCR analysis of VSMCs after transfection with an ASO targeting exon 9, showing a band at ~ 300 bp, corresponding to the expected fragment size after exon 9 skipping, which was confirmed to be a correct exon 9 skip with Sanger sequencing. ( B ) Guide RNAs targeting NOTCH3 introns 8 and 9 were used to delete exon 9 from the genomic DNA of HEK293 cells. RT-PCR analysis confirmed exon 9 exclusion at the RNA level. ( C ) Guide RNAs targeting intron 3 and intron 5 were used to delete exon 4 and exon 5 from the genomic DNA from control (left lane) and CADASIL patient-derived VSMCs (right lane), which was confirmed with Sanger sequencing. FL, full length; −RT, condition without reverse transcriptase; Δex9, PCR product lacking exon 9; Δex4–5, PCR product lacking exon 4–5; arrowhead, aspecific genomic PCR product.

Article Snippet: In short, cells were either fixed and permeabilized with 4% paraformaldehyde and 0.1% Triton X-100 or only fixed without permeabilization using 4% paraformaldehyde and stained with a mouse anti-NOTCH3 ECD antibody (clone 1E4, Millipore, dilution 1:10 000).

Techniques: CRISPR, In Vitro, Reverse Transcription Polymerase Chain Reaction, Transfection, Sequencing, Control, Derivative Assay, Reverse Transcription