anti notch1 extracellular domain ecd antibody  (R&D Systems)

Journal: JID Innovations

Article Title: Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin

doi: 10.1016/j.xjidi.2025.100444

Figure Lengend Snippet: The inhibition of the Notch pathway increases miR-30a activity. (a ) Representative images of reconstructed epidermises obtained using RIFES miR-30a-3p or miR-30a-5p primary keratinocytes. The green fluorescence corresponds to GFP, and the blue fluorescence corresponds to nuclei labeled with DAPI. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. ( b ) Immunofluorescence labeling of Notch 1 (denoted as NICD) in a skin biopsy (female abdominal skin, young adult). The red fluorescence corresponds to Notch1, the green fluorescence corresponds to loricrin, and the blue fluorescence corresponds to nuclei labeled with DAPI. Bar = 50 mm. ( c ) Western blot analysis of Notch1 (denoted as NICD), cleaved Notch1, and actin in HPKs treated or not by DAPT. ( d ) HEY1 transcript relative expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). ( e ) Response of the RIFES miR-30a-3p or -5p HPKs to DAPT treatment. The graph corresponds to the quantification of the GFP fluorescence by HCS after DAPT or control treatment (box plot with Tukey whiskers, n = 4, ∗ P < .05 t -test P -value). Representative images of cells are shown (Merge image: GFP plus DAPI). ( f ) Western blot analysis of GFP and actin expression in lentiRIFES/miR-30a-3pT or 5pT HPKs after DAPT treatment. ( g ) Reconstructed epidermises were obtained using lentiRIFES/miR-30a-3pT or 5pT keratinocytes. The RHEs were treated or not by the DAPT compound. Representative images of GFP fluorescence or K10 immunofluorescence are shown. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. A small area of the images surrounded by a frame is shown at higher magnification. ( h ) miR-30a-3p or miR-30a-5p transcript expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). HCS, high-content screening; HPK, human primary keratinocyte; K10, keratin 10; RHE, reconstructed human epidermis.

Article Snippet: Membranes were blocked with 10% nonfat dry milk for 1 hour and incubated with primary antibodies against NOTCH 1 (number 3608, Cell Signaling Technology), cleaved NOTCH-1 (number 4147, Cell Signaling Technology), c-MYC, or β-actin (ab8227, Abcam) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Fluorescence, Labeling, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR, Control, High Content Screening

Journal: JID Innovations

Article Title: Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin

doi: 10.1016/j.xjidi.2025.100444

Figure Lengend Snippet: The inhibition of the Notch pathway increases miR-30a activity. (a ) Representative images of reconstructed epidermises obtained using RIFES miR-30a-3p or miR-30a-5p primary keratinocytes. The green fluorescence corresponds to GFP, and the blue fluorescence corresponds to nuclei labeled with DAPI. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. ( b ) Immunofluorescence labeling of Notch 1 (denoted as NICD) in a skin biopsy (female abdominal skin, young adult). The red fluorescence corresponds to Notch1, the green fluorescence corresponds to loricrin, and the blue fluorescence corresponds to nuclei labeled with DAPI. Bar = 50 mm. ( c ) Western blot analysis of Notch1 (denoted as NICD), cleaved Notch1, and actin in HPKs treated or not by DAPT. ( d ) HEY1 transcript relative expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). ( e ) Response of the RIFES miR-30a-3p or -5p HPKs to DAPT treatment. The graph corresponds to the quantification of the GFP fluorescence by HCS after DAPT or control treatment (box plot with Tukey whiskers, n = 4, ∗ P < .05 t -test P -value). Representative images of cells are shown (Merge image: GFP plus DAPI). ( f ) Western blot analysis of GFP and actin expression in lentiRIFES/miR-30a-3pT or 5pT HPKs after DAPT treatment. ( g ) Reconstructed epidermises were obtained using lentiRIFES/miR-30a-3pT or 5pT keratinocytes. The RHEs were treated or not by the DAPT compound. Representative images of GFP fluorescence or K10 immunofluorescence are shown. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. A small area of the images surrounded by a frame is shown at higher magnification. ( h ) miR-30a-3p or miR-30a-5p transcript expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). HCS, high-content screening; HPK, human primary keratinocyte; K10, keratin 10; RHE, reconstructed human epidermis.

Article Snippet: Membranes were blocked with 10% nonfat dry milk for 1 hour and incubated with primary antibodies against NOTCH 1 (number 3608, Cell Signaling Technology), cleaved NOTCH-1 (number 4147, Cell Signaling Technology), c-MYC, or β-actin (ab8227, Abcam) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Fluorescence, Labeling, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR, Control, High Content Screening

Journal: bioRxiv

Article Title: Absence of EOGT Precludes Defective Development in Fringe-null Mouse Intestine

doi: 10.64898/2026.02.10.705133

Figure Lengend Snippet: O-Glycans of EGF repeats and consequences of their removal. (A) Diagram depicting the EGF repeats of NOTCH1, NOTCH2, DLL1 and DLL4 extracellular domains. The consensus sequence recognized by the glycosyltransferase that transfers a Fuc, Glc or GlcNAc to Ser/Thr in an EGF repeat is given on the left and the sequence of sugars that may occur at each site is given on the right. The Controls column shows the full sugar extension that may occur at each site with the enzyme(s) that would be responsible for transfer [ , - ]. The other columns show the sequences remaining after deletion of Lfng alone ( Lfng cKO) or all three Fringes together (LMR tKO) or all three Fringes in an Eogt -null background (ELMR qKO). Parentheses indicate potential additions. Shaded sugars in Lfng cKO reflect potential addtion by MFNG and/or RFNG. (B, C) Body weight (B) and small intestine (SI) length (C) in control and experimental mice (n ≥ 7 mice per group). P values from one-way ANOVA followed by Tukey’s multiple comparisons test ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were assessed for NOTCH1 surface expression with anti-NOTCH1 extracellular domain (ECD) antibody (Ab AF5267; R&D Systems, Inc., Minneapolis, MN) and binding of Notch ligands DLL1-Fc (R&D Systems, Inc.) and DLL4-Fc (Acro Biosystems, Newark, DE).

Techniques: Sequencing, Control

Journal: bioRxiv

Article Title: Absence of EOGT Precludes Defective Development in Fringe-null Mouse Intestine

doi: 10.64898/2026.02.10.705133

Figure Lengend Snippet: Notch ligand binding to ISC in crypts. (A-C) Flow cytometry profiles of anti-NOTCH1 ECD Ab, DLL1-Fc and DLL4-Fc binding to ISC from Control, LMR tKO and ELMR qKO crypts. Pink profiles show Ab to NOTCH1 ECD or Notch ligand-Fc binding, blue profiles show 2° antibody non-specific binding. (D) MFI for Ab to NOTCH1 ECD, DLL1-Fc and DLL4-Fc binding to LMR tKO and ELMR qKO normalized to MFI of Control in each of six experiments performed over 6 days. MFI values varied between experiments so each experiment was analyzed separately. One-way ANOVA followed by Tukey’s multiple comparisons test was used to determine P values.

Article Snippet: Cells were assessed for NOTCH1 surface expression with anti-NOTCH1 extracellular domain (ECD) antibody (Ab AF5267; R&D Systems, Inc., Minneapolis, MN) and binding of Notch ligands DLL1-Fc (R&D Systems, Inc.) and DLL4-Fc (Acro Biosystems, Newark, DE).

Techniques: Ligand Binding Assay, Flow Cytometry, Binding Assay, Control

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Dietary nitrate inhibits the Notch pathway overactivated by ethanol in SD rats. (A) Bubble plot showing the top 20 significantly enriched KEGG pathways. The abscissa is the ratio of the number of differential genes annotated to the KEGG pathway to the total number of differential genes, and the ordinate is the KEGG pathway. (B) Representative immunoblotting band of Notch1, Nicd, and Rbpj protein in gastric mucosal tissue. (C–E) Analyses of immunoblotting band gray value in (B). (F) GSEA analysis of Notch signaling pathway between ulcer and control groups. (G) GSEA analysis of Notch signaling pathway between ulcer + Nit and ulcer groups. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. KEGG, Kyoto Encyclopedia of Genes and Genomes; Nicd, intracellular structural domain; Rbpj, recombination signal binding protein for immunoglobulin kappa J region; GSEA, gene set enrichment analysis; RNA seq, RNA sequencing; SD,D standard deviation.

Article Snippet: Following incubation with primary NICD (NBP1‐48289; Novus Biologicals, USA) and RBPJ (720219; Thermo, USA) antibodies, a pair of Duolink PLA probes—anti‐mouse MINUS (DUO92004; Merck) and anti‐rabbit PLUS (DUO92002; Merck)—were applied for 60 min. Ligase was added for 30 min followed by amplification of signal for 100 min using polymerase.

Techniques: Western Blot, Control, Binding Assay, RNA Sequencing, Standard Deviation

Journal: MedComm

Article Title: Nitrate Enhances Gastric Mucosa Defense and Repair Process in Ethanol‐Induced Gastric Ulcer Rats via the Notch–Tff2 Pathway

doi: 10.1002/mco2.70628

Figure Lengend Snippet: Nitrate functions by Notch pathway inhibition positively transcripting TFF2 in vitro. (A) Diagram of the wild‐type and mutant sequences for the three predicted RBPJ binding sites within the 251 bp TFF2 promoter probes. (B) Representative electrophoretic mobility shift assay (EMSA) autoradiograph. Hot probe is the biotin‐labeled wild‐type oligonucleotides of the truncated TFF2 promoter containing the binding motif; Mutant probe is the labeled oligonucleotides sequence with nucleotides mutated. The cold probe is nonlabeled competitive wild‐type probes (100 and 50 that of the concentrations). The shifted bands are indicated by arrows, which suggested the formation of DNA–protein complexes (lane 2, 3, 7). The super shifted bands indicated the formation of DNA–protein–antibody complexes (lane 3, 7). “+” and “−” represent presence and absence, respectively. (C) Relative TFF2 promoter (Full, Mut1, Mut2, and Mut3) luciferase activity was detected by DLR assays in RBPJ overexpressed and normal‐expressed GES‐1 cells. (D) A schematic diagram showing the location of RBPJ putative binding regions on the TFF2 promoter. (E) RT‐qPCR analysis of TFF2 binding site expression of GES‐1 cells. Target site expression in RBPJ‐treated groups was normalized to IgG negative control groups and expressed as fold change relative to the IgG groups. (F) IF staining of NICD (pink) and DAPI (blue). Scale bar = 50 µm. (G) IF analysis of MFI of nuclear NICD in positive cells. (H) Representative immunoblotting band of Notch signaling pathway in GES‐1 cells. (I–K) Analyses of immunoblotting band gray value of (H). (L) RT‐qPCR analysis of TFF2 mRNA expression of DMSO/DAPT treated GES‐1 cells. Target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the DMSO vehicle group. (M) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD deprived GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector + DMSO group. (N) RT‐qPCR analysis of TFF2 mRNA expression in RBPJ overexpressed and NICD‐RBPJ overexpressed GES‐1 cells. The target gene expression was normalized to GAPDH mRNA and expressed as fold change relative to the vector1 + vector2 group. (O) Representative image of PLA of NICD–RBPJ proximity. Each red dot represents a positive signal of NICD–RBPJ interaction and nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns denotes no significance. Nit, nitrate; EtOH, ethanol; GES‐1, human gastric epithelial; NICD, intracellular structural domain; RBPJ, recombination signal binding protein for immunoglobulin kappa J region; EMSA, electrophoretic mobility shift assay; TFF2, trefoil factor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; Yhhu‐3792, N2‐(4‐isopropylphenyl)‐5‐(3‐methoxyphenoxy) quinazoline‐2,4‐diamine; Luc, luciferase; SD, standard deviation; DLR, dual‐luciferase report; PLA, proximity ligation assay.

Article Snippet: Following incubation with primary NICD (NBP1‐48289; Novus Biologicals, USA) and RBPJ (720219; Thermo, USA) antibodies, a pair of Duolink PLA probes—anti‐mouse MINUS (DUO92004; Merck) and anti‐rabbit PLUS (DUO92002; Merck)—were applied for 60 min. Ligase was added for 30 min followed by amplification of signal for 100 min using polymerase.

Techniques: Inhibition, In Vitro, Mutagenesis, Binding Assay, Electrophoretic Mobility Shift Assay, Autoradiography, Labeling, Sequencing, Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Negative Control, Staining, Western Blot, Targeted Gene Expression, Plasmid Preparation, Standard Deviation, Proximity Ligation Assay

Journal: Journal of Advanced Research

Article Title: An injectable nano-hydroxyapatite-incorporated hydrogel with sustained release of Notoginsenoside R1 enhances bone regeneration by promoting angiogenesis through Notch1/Akt signaling

doi: 10.1016/j.jare.2025.05.025

Figure Lengend Snippet: (A) The angiogenic-related gene expression quantified by qRT-PCR at day 4, day 7 and day 10. (B) The angiogenic proteins of VEGF and VEGFR-2 analyzed by western blot. (C)semiquantitative analysis of VEGF and VEGFR-2 protein expression. (D) The critical markers Notch1, Hes1 involved in the Notch 1 pathway analyzed by western blot and their semiquantitative analysis (E).(F)Western blot and semiquantitative analysis (G) of Akt protein expression.(H)The gene expression of Akt and VEGF under NGR1 stimulation with Notch 1 inhibition. (I) The gene expression of Notch 1 and VEGF under NGR1 stimulation with Akt inhibition. (J) Western blot and semiquantitative analysis (K) of Akt protein expression induced by NGR1 under Notch 1 inhibition. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

Article Snippet: Subsequently, Notch 1 inhibitor (HY-1302, MCE, USA) and Akt inhibitor (HY-10358, MCE, USA) were applied to evaluate their effects on angiogenic-related gene expression.

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Expressing, Inhibition