notch3 rabbit mab  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Receptor stoichiometry predicts artery-typical vulnerability to altered Notch signaling during smooth muscle differentiation

doi: 10.1101/2025.05.05.652186

Figure Lengend Snippet: Notch2 and Notch3 act as functional analogs during Jagged1-mediated SMC differentiation, but Notch2 facilitates the early transcriptional response. (A) Schematic description of the experimental setup and the analysis strategy for the RNAseq experiment. (B) Notch2 and Notch3 protein levels in SMCs 48h post-transfection with the corresponding siRNAs. (C) Genes significantly regulated in SMCs on immobilized J1-Fc at 4h and 24h (FC>2, FDR>0.05) based on RNAseq. The top 10 up- or downregulated genes are annotated (n=3 independent experiments). (D) Heatmaps clustered by Ward’s method showing the z-scores of genes regulated in SMCs on immobilized J1-Fc at 4h and 24h time points across cells treated with control, Notch2 or Notch3 siRNAs (n=3 independent experiments). Gene clusters were considered as follows: Cluster c; upregulated more by Notch2 and less by Notch3, cluster d; upregulated by both Notch2 and Notch3, and Cluster e; downregulated both by Notch2 and Notch3. (E) Behavior of the gene clusters upon different treatments: genes upregulated on immobilized J1-Fc at 4h, 24h cluster d, and 24h cluster e, respectively. (F) Lists of representative genes related to SMC differentiation and extracellular matrix production upregulated on J1-Fc (Cluster d) and genes related to proliferation downregulated on J1-Fc (Cluster e). (G-I) Expression of genes representative of Notch signaling activity, contractile differentiation, and cell cycle arrest (mean±SD). (J) Gene ontology analysis of the clusters from 24h as listed by the “clusterprofiler” package.

Article Snippet: The primary antibodies used: Notch2 1:1000 (Cell Signaling Cat. #5732), Notch3 1:1000 (Cell Signaling Cat. #5276), Jagged1 1:1000 (Cell Signaling Cat. #2620), α-SMA 1:1000(Cell Signaling Cat. #19245), Pdgfrβ 1:1000 (Cell Signaling Cat. #3169), β-tubulin 1:1000 (Cell Signaling Cat. #86298), His-Tag 1:1000 (Cell Signaling Cat. #2365), M2-Flag 1:1000 (Merck, Cat. #F1804), smad2/3 1:200 (Santa Cruz Cat. #sc-133098), and phospho-smad2 1:1000 (Cell Signaling Cat. #3108), β-actin 1:10000 (Merck Cat. #A1978).

Techniques: Functional Assay, Transfection, Control, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Receptor stoichiometry predicts artery-typical vulnerability to altered Notch signaling during smooth muscle differentiation

doi: 10.1101/2025.05.05.652186

Figure Lengend Snippet: The Jagged1-Notch2 axis upregulates Notch3 for the progression of the differentiation program. Expression of NOTCH2 and NOTCH3 mRNA across the groups during the RNAseq experiment (mean±SD, n=3 independent experiments). The DAPT washout experiment setup was used for the analysis of the Notch protein levels during the J1-Fc mediated differentiation. Schematic description of the probing method used for detecting the Notch receptor products. Using antibodies against epitopes on the C-terminal end of the Notch receptor peptides allows for the detection of fragments, namely, Pre-Notch (non-membranous Notch protein that has not gone through S1-cleavage), NTM (S1-cleaved Notch transmembrane- intracellular domain), and NEXT (S2-cleaved Notch extracellular-truncated intracellular domain). In reducing lysis conditions, the NTM and the NECD (Notch extracellular domain) that are held together via physical bonds dissociate. Thus, here the NTM and its products were used to assess the levels of the mature membranous Notch receptor. (D, E) The immunoblot analysis (D) and the quantified signal from the indicated proteins relative to the protein loading (E) in SMCs plated on immobilized J1-Fc over five days. The dotted lines represent log2 fold change (mean±SEM, n=3 independent experiments, one-way repeated measures ANOVA, and Dunnet’s test for multiple comparisons. Pairwise comparisons with p values smaller than 0.1 are indicated on the plot). (F, G) The setup used for the DAPT washout experiment to assess the impact of Notch2 or Notch3 knockdown on Notch signaling in SMCs plated on immobilized J1-Fc and the qPCR analysis (E) for the indicated mRNA levels. The dotted lines represent relative expression (mean±SEM, n=3 independent experiments (n=2 for 72h). Two-way ANOVA and Tukeýs test for multiple comparisons were performed individually for different time points. Pairwise comparisons between J1-Fc-treated samples with p values smaller than 0.1 are indicated on the plots and color-coded for the groups compared). (H) Schematic description of the working model regarding the temporal relationship between Jagged1, Notch2, and Notch3 and the Notch signaling output over time-based on figures 1,2, and S3.

Article Snippet: The primary antibodies used: Notch2 1:1000 (Cell Signaling Cat. #5732), Notch3 1:1000 (Cell Signaling Cat. #5276), Jagged1 1:1000 (Cell Signaling Cat. #2620), α-SMA 1:1000(Cell Signaling Cat. #19245), Pdgfrβ 1:1000 (Cell Signaling Cat. #3169), β-tubulin 1:1000 (Cell Signaling Cat. #86298), His-Tag 1:1000 (Cell Signaling Cat. #2365), M2-Flag 1:1000 (Merck, Cat. #F1804), smad2/3 1:200 (Santa Cruz Cat. #sc-133098), and phospho-smad2 1:1000 (Cell Signaling Cat. #3108), β-actin 1:10000 (Merck Cat. #A1978).

Techniques: Expressing, Lysis, Western Blot, Knockdown

Journal: bioRxiv

Article Title: Receptor stoichiometry predicts artery-typical vulnerability to altered Notch signaling during smooth muscle differentiation

doi: 10.1101/2025.05.05.652186

Figure Lengend Snippet: Elevated levels of Notch3 are required for transcriptionally meaningful canonical Notch3 signaling due to the weak Notch3-CSL interaction. (A) Schematic representation of the live staining of Notch receptors in the SMCs with labeled soluble J1-Fc. (B) Confocal slices of formalin-fixed SMCs (control, Notch2KD, and Notch3KD) following live staining with J1-Fc (Scale bar=50μm). (C, D) Representative histograms and the bar plots (D) showing the fold change in mean intensity of live cells stained with J1-Fc measured using FACS (mean+SEM, n=3 independent experiments, one-way ANOVA followed by Šídák’s test for multiple comparisons. Pairwise comparisons between J1-Fc-stained samples with p values lower than 0.1 are shown on the plot.). (E, F) Immunoblots produced by C-terminal probing of Notch2 and Notch3 following DAPT washout in SMCs plated on immobilized J1-Fc, and the quantitative analysis of the bands. (F)The receptor cleavage was measured by the ratio of the NEXT (lower band) to the total S1- processed fragment (NEXT+TMIC (higher band)) (mean+SEM, n=3 independent experiments, one-way ANOVA followed by Šídák’s test for multiple comparisons. Pairwise comparisons with p values lower than 0.1 are shown on the plot.). (G, H) CSL-dependent Notch transcriptional activity in HEK 293T cells transfected with increasing amounts of Flag-tagged Notch2 or Notch3 ICD and 12xCSL-luciferase. The luciferase activity is presented as luminescence intensity relative to β-galactosidase activity. The immunoblots show the expression of the NICD-Flag constructs relative to β-tubulin (H). (mean+SEM, n=3 independent experiments. Data are presented as regression curves and circles as replicates.). (I, J) Flag-tagged NICD paralogs were immunoprecipitated from HEK 293T cells co-expressing 6xHis-tagged RBPJκ. The Flag and the 6xHis IP signals were normalized to the input. The normalized 6xHis/Flag signal is presented in the bar plot (J) (mean+SEM, n=3 independent experiments, one-way ANOVA followed by Tukey’s test for multiple comparisons. Pairwise comparisons between NICD paralogs with p values lower than 0.1 are shown on the plot.).

Article Snippet: The primary antibodies used: Notch2 1:1000 (Cell Signaling Cat. #5732), Notch3 1:1000 (Cell Signaling Cat. #5276), Jagged1 1:1000 (Cell Signaling Cat. #2620), α-SMA 1:1000(Cell Signaling Cat. #19245), Pdgfrβ 1:1000 (Cell Signaling Cat. #3169), β-tubulin 1:1000 (Cell Signaling Cat. #86298), His-Tag 1:1000 (Cell Signaling Cat. #2365), M2-Flag 1:1000 (Merck, Cat. #F1804), smad2/3 1:200 (Santa Cruz Cat. #sc-133098), and phospho-smad2 1:1000 (Cell Signaling Cat. #3108), β-actin 1:10000 (Merck Cat. #A1978).

Techniques: Staining, Labeling, Control, Western Blot, Produced, Activity Assay, Transfection, Luciferase, Expressing, Construct, Immunoprecipitation

Journal: bioRxiv

Article Title: Receptor stoichiometry predicts artery-typical vulnerability to altered Notch signaling during smooth muscle differentiation

doi: 10.1101/2025.05.05.652186

Figure Lengend Snippet: Levels of Notch3 predict artery-typical vulnerability to altered Jagged1-Notch2/Notch3 signaling in the SMCs. (A-C) Normalized expression of Notch signaling components and Notch target gene Nrarp in arterial SMCs (A and C) and vascular endothelial cells (B) of different mouse organs extracted from scRNAseq data (Muhl et al. ; aorta, heart, colon, and lung) and (Vanlandewijck et al. ; brain). The circles and the text above the violin plots denote the mean expression. Schematic one-dimensional array of 1D artery model with an endothelial cell and a row of SMCs. The cascade of signaling is initiated by constant Jagged1 emission from the endothelial cell (EC) and SMCs are interacting with their neighboring cells. Heatmaps showing the simulated impact of the initial Jagged1, Notch2, and Notch3 levels on the Notch signaling readout (Notch multiplier) in the 1D hybrid arterial wall. 2D projection of the predicted ranges of Notch multiplier in arterial SMCs of different organs with respect to their stoichiometrically scaled levels of Jagged1, Notch2, and Notch3 based on A, and in a Notch2 knockout scenario.

Article Snippet: The primary antibodies used: Notch2 1:1000 (Cell Signaling Cat. #5732), Notch3 1:1000 (Cell Signaling Cat. #5276), Jagged1 1:1000 (Cell Signaling Cat. #2620), α-SMA 1:1000(Cell Signaling Cat. #19245), Pdgfrβ 1:1000 (Cell Signaling Cat. #3169), β-tubulin 1:1000 (Cell Signaling Cat. #86298), His-Tag 1:1000 (Cell Signaling Cat. #2365), M2-Flag 1:1000 (Merck, Cat. #F1804), smad2/3 1:200 (Santa Cruz Cat. #sc-133098), and phospho-smad2 1:1000 (Cell Signaling Cat. #3108), β-actin 1:10000 (Merck Cat. #A1978).

Techniques: Expressing, Knock-Out

Journal: bioRxiv

Article Title: Receptor stoichiometry predicts artery-typical vulnerability to altered Notch signaling during smooth muscle differentiation

doi: 10.1101/2025.05.05.652186

Figure Lengend Snippet: Artery-typical elevated Notch3 expression in the developing SMCs can compensate for Notch2 depletion in the zebrafish larvae (Created with Biorender). (A) Schematic description of the vascular tree of the zebrafish larvae, and the position of the DA, the PCS, and the BCA (Created using Biorender). (B, C) In situ RNA HCR was performed with probes against notch2 and notch3 in 3dpf zebrafish larvae bearing a pdgfrb reporter. Maximum intensity projections of five slice-thick confocal sections of the arteries from the cerebral base (B) and the dorsal aorta (C) given with dashed lines loosely marking the arterial regions based on the pdgfrb signal are shown (Scale bars=20 μm) ( DA: dorsal aorta, BCA: basal communicating artery, PCS: posterior communicating segment, CaDI: caudal division of the internal carotid artery, MtA: metencephalic artery) UMAP projection of the SMCs color-coded based on the Seurat clusters and the developmental stage from the Zebrahub mesoderm scRNAseq dataset . Cluster annotations were based on known markers of vascular and non-vascular SMCs as well as notch3 expression (Please see Figure 5S). The vascular SMCs were annotated based on the time point and notch3 expression status (high or low). (E, F) Normalized expression of notch3, the general mural marker pdgfrb, the anteroposterior position-related homeobox gene hoxc1a; and (F) the normalized expression of the Notch signaling components in the indicated vascular SMC clusters at developmental stages relevant for mural cell emergence. (G, H) The embryos bearing a contractility reporter (tagln:GFP) or the endothelial reporter (kdrl:mCherry) were injected with 10ng control, notch2 or notch3 morpholinos. The orthogonal view of the surface-rendered light sheet images of the indicated arteries from the 102 hpf (G, the BCA/PCS, scale bar=100μm and H, the DA and the ISV, scale bar=200μm). The number of tagln+ objects associated with the endothelium are shown in the bar plots (H) (n=3 or 4 embryos per morphant group, one-way ANOVA followed by Tukeýs test for multiple comparisons. Pairwise comparisons with p values smaller than 0.1 are shown on the plots.).

Article Snippet: The primary antibodies used: Notch2 1:1000 (Cell Signaling Cat. #5732), Notch3 1:1000 (Cell Signaling Cat. #5276), Jagged1 1:1000 (Cell Signaling Cat. #2620), α-SMA 1:1000(Cell Signaling Cat. #19245), Pdgfrβ 1:1000 (Cell Signaling Cat. #3169), β-tubulin 1:1000 (Cell Signaling Cat. #86298), His-Tag 1:1000 (Cell Signaling Cat. #2365), M2-Flag 1:1000 (Merck, Cat. #F1804), smad2/3 1:200 (Santa Cruz Cat. #sc-133098), and phospho-smad2 1:1000 (Cell Signaling Cat. #3108), β-actin 1:10000 (Merck Cat. #A1978).

Techniques: Expressing, In Situ, Marker, Injection, Control

Journal: bioRxiv

Article Title: Receptor stoichiometry predicts artery-typical vulnerability to altered Notch signaling during smooth muscle differentiation

doi: 10.1101/2025.05.05.652186

Figure Lengend Snippet:

Article Snippet: The primary antibodies used: Notch2 1:1000 (Cell Signaling Cat. #5732), Notch3 1:1000 (Cell Signaling Cat. #5276), Jagged1 1:1000 (Cell Signaling Cat. #2620), α-SMA 1:1000(Cell Signaling Cat. #19245), Pdgfrβ 1:1000 (Cell Signaling Cat. #3169), β-tubulin 1:1000 (Cell Signaling Cat. #86298), His-Tag 1:1000 (Cell Signaling Cat. #2365), M2-Flag 1:1000 (Merck, Cat. #F1804), smad2/3 1:200 (Santa Cruz Cat. #sc-133098), and phospho-smad2 1:1000 (Cell Signaling Cat. #3108), β-actin 1:10000 (Merck Cat. #A1978).

Techniques: