polyclonal sheep anti notch3 ecd (R&D Systems)
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polyclonal sheep anti notch3 ecd
Polyclonal Sheep Anti Notch3 Ecd, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/polyclonal sheep anti notch3 ecd/product/R&D Systems
Average 93 stars, based on 46 article reviews
Polyclonal Sheep Anti Notch3 Ecd, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/polyclonal sheep anti notch3 ecd/product/R&D Systems
Average 93 stars, based on 46 article reviews
polyclonal sheep anti notch3 ecd - by Bioz Stars,
2026-03
93/100 stars
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1) Product Images from "Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL"
Article Title: Heterogeneity of NOTCH3 activation mechanisms uncovers therapeutic potential for targeted therapies for CADASIL
Journal: bioRxiv
doi: 10.1101/2024.11.17.623993
Figure Legend Snippet: (A) Modular structure of NOTCH3, SP indicates signal peptide, red shaded EGF-like modules indicate core ligand binding region, LNR indicates Lin12-Notch repeats, NRR is the negative regulatory region, S1, S2 and S3 are proteolytic cleavage sites, HD-N and HD-C are the heterodimer interface region. RAM and Ankyrin repeat region (Ank) are motifs involved in binding CSL transcription factors, PEST domain is involved in ubiquitin-dependent turnover of the ICD. Location of mutants used in this study are indicated by arrows. Coloured bars indicate locations of regions of NOTCH3 in which epitopes used in this study are located. (B-D) AlphaFold2 predictions of WT NOTCH3 EGF modules 2 (B) , 5 (C) and 11 (D) , and mutant substitutions introduced using the SDM programme. Location of the new Cys residue in R90C indicated in yellow, and other substitutions that replace as cysteine residues are shown in magenta. (E-G) Surface views of the structures from (B-D) depicted as a surface mesh. The introduced cysteine in R90C is surface exposed (E) , but the free cysteines in C212Y and C455R are buried within the module structure. ΔΔG values indicate predicted decreases in stability. (H-K) Expressed WT NOTCH3 (H) and CADASIL mutants (I-K) do not show strong accumulation in the ER when expressed in hTert-RPE1 cells. (L, M) scoring of NOTCH3 localisation in KDEL-positive ER, by % area of ER occupied (L) and by % of total NOTCH3 staining intensity (K) in z-sections through the cytoplasmic region. R90C shows a small increase in %NOTCH3 which is localised to the ER. * indicates P<0.05 by student t-test, error bars SEM, n=10. (N) Antibody surface labelling of NOTCH3 localisation on non-permeabilised cells at time 0 and 60 minutes during a live cell anti-ECD uptake assay. Similar time zero surface distributions, and subsequent surface depletion of WT and CADASIL mutants can be seen.
Techniques Used: Ligand Binding Assay, Binding Assay, Mutagenesis, Residue, Staining
Figure Legend Snippet: (A-C) Schematic view of pulse chase labelling protocol. (D-G) WT NOTCH3 localisation in permeabilised cells after antibody uptake at 0 (D) , 15 (E) , 30 (F) , and 60 (G) minutes. Little surface-labelled NOTCH3 is transported to the early endosome by 15 minutes chase but is evident in EEA1 positive endosomes by 30 and 60 minutes. Presence of both full-length and ECD-only staining suggests ECD-shedding can occur by the time NOTCH3 is localised to the EEA1-marked endosome. (H-J) CADASIL mutant NOTCH3 localisation after 60-minute chase shows the mutant proteins labelled at the cell surface also become localised to the EEA1-positive endosome. (K, L) After 60-minute chase surface, little labelled NOTCH3 ECD has reached the CD63-positive late endosome while ICD is present. However full-length NOTCH3 and ECD-only localisations are found adjacent to CD63-marked organelles.
Techniques Used: Pulse Chase, Staining, Mutagenesis
Figure Legend Snippet: (A-D) Immunolocalisation of NOTCH3 with anti-ECD and anti-ICD compared with EEA1-marked endosomes in fixed and permeabilised cells of WT (A) and CADASIL mutants ( B-D) . (E) Quantitation of NOTCH3 localisation in the early endosome, showing % endosomal-located NOTCH3 puncta which either full-length NOTCH3, ECD-only, or ICD-only staining. R90C is distinguished from WT and other mutants by a greater proportion of ICD-only puncta compared to ECD or full-length. For WT, n=91 NOTCH3 puncta from198 endosomes scored. For R90C, n=139 NOTCH3 puncta from 180 endosomes score. For C212Y, n=140 NOTCH3 puncta from 240 endosomes scored. For C455R, n=164 NOTCH3 puncta from 302 endosomes scored. Minimum of 7 cells scored for each construct. Data obtained from z sections from at least 7 cells. (F) Full-length NOTCH3, or ECD-only puncta located within or adjacent to Rab11-positive endosomes. (G, H) In CD63 positive endosomes (G) and LAMP1-positive lysosomes (H) , NOTCH3 localisation is predominantly as ICD-only puncta, quantified in (I, J) . Error bars in I and J are SEM, n=11 and n=7 respectively. Statistical significance by student t-test.
Techniques Used: Quantitation Assay, Staining, Construct
Figure Legend Snippet: (A) Schematic diagram illustrating alternative explanations for visualisation of separated ECD and ICD-positive puncta, including: ECD shedding by S2 cleavage; epitope masking; fragmentation to remove terminal epitopes; non-specific staining. (B) Colocalisation of two different ECD epitopes compared to ICD ab23426 . ICD-stained puncta can be observed which lack both ECD epitopes. (C) Colocalisation of two different ICD epitopes compared to anti-ECD 1E . ECD-only spots are present which lack both ICD epitopes. (D, E) Quantification of the colocalisation between the epitopes used in B, C. In D * indicates P<0.05 compared with double ECD epitope colocalisation by student t-test. Error bars represent SEM, n=5 (D) , n=14 (E) . (F-H) Both ICD epitopes are located to the nucleus in NOTCH3 transfected cells but not the ECD epitope.
Techniques Used: Staining, Transfection
Figure Legend Snippet: (A) EEA1-positive early endosomes of MCF7 cells costained with anti-ECD, and ICD. Arrowheads indicate endosomes shown enlarged in insets which have separate localisation of ECD and ICD puncta. (B) CD63-positive late endosomes costained with anti-ECD, and ICD. Boxed region is enlarged in inset showing late endosomes predominantly contain ICD-only puncta. (C) After treatment of MCF7 cells with metalloprotease inhibitor BB94, more full-length NOTCH3 staining is observed in the late endosomes, colocalised with CD63. (D) Treatment of cells with gamma-secretase inhibitor DAPT does not alter localisation of ECD in the late endosome. (E-G) Quantification of colocalization of ECD vs ICD (E) , ECD vs CD63 (F) , and ICD vs CD63 ( G) . Error bars, SEM, control DMSO-only treated cells n=10, BB94 n=18, DAPT n=12. * indicates p<0.05 by student t-test compared to control.
Techniques Used: Staining, Control
Figure Legend Snippet: (A) Schematic figure illustrating luciferase reporter assay methodology. (B) WT and CADASIL mutant NOTCH3 signalling after transfection into hTERT-RPE1 cells compared to empty vector (EV) control. * indicates p<0.05 compared to WT NOTCH3 by student t-test. Error bars SEM, minimum of n=4. (C-F) NOTCH3 signalling after treatment of transfected cells with TRPML inhibitor (ML-Sl1), gamma-secretase inhibitor (RO 4929097 ), or metalloprotease inhibitor (BB94) for WT (C) , R90C (D) , C212Y (E) , and C455R (F) * indicates p<0.05 compared with control by student t-test. Error bars are SEM, minimum of n=3.
Techniques Used: Luciferase, Reporter Assay, Mutagenesis, Transfection, Plasmid Preparation, Control
