antibodies against notch 1  (Cell Signaling Technology Inc)

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: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of Notch1 intracellular domain (N1ICD) expression on the expression of total Orai1 and Orai1α and Orai1β isoforms in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were transfected with myc-N1ICD fragment (myc-N1ICD) or empty vector (Control) and 24 h later cells were lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with anti-myc or anti-Orai1 antibody, as described in Materials and Methods. Membranes were reprobed with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 10, 13,10, 7 and 11 separate experiments for MCF10A, MDA-MB-231, BT20, MCF-7 and T47D, respectively. ( F – H ) Bar graphs represent the quantification of Orai1 ( F ), Orai1α ( G ) or Orai1β ( H ) protein expression as fold change over the level in mock-transfected cells (Control) and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05 and ** p < 0.01.

Article Snippet: Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, SDS Page, Western Blot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of Notch1 intracellular domain (N1ICD) expression on the expression of STIM1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were transfected with myc-N1ICD fragment (myc-N1ICD) or empty vector (Control) and 24 h later cells were lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with anti-myc, anti-STIM1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. * Non-specific band. n = 9, 13, 8, 11 and 10 separate experiments for Orai3 and 4, 6, 4, 3 and 5 separate experiments for STIM1 in MCF10A, MDA-MB-231, BT20, MCF-7 and T47D, respectively. ( F , G ) Bar graphs represent the quantification of Orai3 ( F ) or STIM1 ( G ) protein expression as fold change over the level in mock-transfected cells (Control) and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05 and ** p < 0.01.

Article Snippet: Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, SDS Page, Western Blot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of Jagged-1 in the expression of Orai1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were stimulated with 50 µM Jagged-1 (Jag-1) or the vehicle (Mock) for 48 h and lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-Notch1, anti-Orai1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 5–9 separate experiments. ( F – H ) Bar graphs represent the quantification of protein expression as fold change over the level in vehicle-transfected cells and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05 and ** p < 0.01.

Article Snippet: Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific).

Techniques: Expressing, SDS Page, Western Blot, Control, Transfection, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of DAPT in the expression of Orai1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were treated with 20 µM DAPT or the vehicle (DMSO) for 24, 48–72 h and lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-Notch1, anti-Orai1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 4–6 separate experiments. Bar graphs represent the quantification of protein expression as fold change over the level in vehicle-treated cells and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific).

Techniques: Expressing, SDS Page, Western Blot, Control, MANN-WHITNEY

Journal: Regenerative Therapy

Article Title: Extracellular vesicles-delivered circDB promotes ischemic muscle repair through the miR-34a/USP7/Notch1 signaling pathway

doi: 10.1016/j.reth.2025.08.009

Figure Lengend Snippet: USP7 directly interacts with and stabilizes Notch1 protein. (A) Predicted structures of USP7 and Notch1 using AlphaFold2. (B) STRING-based protein-protein interaction network showing the association between USP7 and Notch1. (C, D) Co-immunoprecipitation (Co-IP) confirming reciprocal binding between USP7 and Notch1 in C2C12 cells (n = 4). (E) Silencing circDB in C2C12 cells for 40 h reduces USP7–Notch1 interaction and increases Notch1 ubiquitination, as shown by Co-IP (n = 3).

Article Snippet: Membranes were incubated with primary antibodies against USP7 (#sc-376912), Notch1 (#sc-376403), CD9 (#sc-13118), GAPDH (#sc-32233; all from Santa Cruz Biotechnology), and K48-Ub (#8081; Cell Signaling Technology).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Ubiquitin Proteomics

Journal: Regenerative Therapy

Article Title: Extracellular vesicles-delivered circDB promotes ischemic muscle repair through the miR-34a/USP7/Notch1 signaling pathway

doi: 10.1016/j.reth.2025.08.009

Figure Lengend Snippet: UMSC-EVs promote USP7 and Notch1 expression via circDB in hindlimb ischemia . (A, B) Western blot (A, n = 4) and qRT-PCR (B, n = 6) analyses showing decreased USP7 and Notch1 expression in ischemic muscle. (C) Western blot analysis of USP7 and Notch1 in C2C12 cells transfected with control or circDB siRNA under hypoxia (n = 4). (D) EdU proliferation assay in C2C12 cells post circDB knockdown under hypoxia (n = 5); blue: Hoechst, red: EdU (scale bar: 100 μm). (E, F) Western blot (E, n = 4) and qRT-PCR (F, n = 4) analyses of USP7 and Notch1 in ischemic mice treated with PBS, NC-EVs, or si-EVs. (G) Fluorescence microscopy showing internalization of PKH26-labeled UMSC-EVs by C2C12 cells (n = 5). (H) EdU staining of C2C12 cells after co-culture with PBS, NC-EVs, or si-EVs (n = 4); blue: Hoechst, red: EdU (scale bar: 100 μm). Data are shown as mean ± SEM.

Article Snippet: Membranes were incubated with primary antibodies against USP7 (#sc-376912), Notch1 (#sc-376403), CD9 (#sc-13118), GAPDH (#sc-32233; all from Santa Cruz Biotechnology), and K48-Ub (#8081; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Proliferation Assay, Knockdown, Fluorescence, Microscopy, Labeling, Staining, Co-Culture Assay

Journal: Regenerative Therapy

Article Title: Extracellular vesicles-delivered circDB promotes ischemic muscle repair through the miR-34a/USP7/Notch1 signaling pathway

doi: 10.1016/j.reth.2025.08.009

Figure Lengend Snippet: USP7 inhibition disrupts circDB-mediated promotion of Notch1 expression and cell proliferation. (A, B) Western blot (A, n = 3) and qRT-PCR (B, n = 3) analysis of USP7 and Notch1 in hypoxia-treated C2C12 cells exposed to PBS, NC-EVs, or si-EVs. (C) EdU assay of proliferating C2C12 cells treated with NC-EVs, si-EVs, or EVs + USP7 inhibitor GNE-6640 (n = 5); blue: Hoechst, red: EdU (scale bar: 100 μm). (D) Western blot analysis of Notch1 protein levels in the same groups (n = 4). Data are presented as mean ± SEM.

Article Snippet: Membranes were incubated with primary antibodies against USP7 (#sc-376912), Notch1 (#sc-376403), CD9 (#sc-13118), GAPDH (#sc-32233; all from Santa Cruz Biotechnology), and K48-Ub (#8081; Cell Signaling Technology).

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, EdU Assay

Journal: Regenerative Therapy

Article Title: Extracellular vesicles-delivered circDB promotes ischemic muscle repair through the miR-34a/USP7/Notch1 signaling pathway

doi: 10.1016/j.reth.2025.08.009

Figure Lengend Snippet: circDB acts as a molecular sponge for miR-34a, upregulating USP7 expression . (A) Bioinformatics prediction (CircBank) of binding sites between circDB and miR-34a. (B–D) Expression levels of miR-34a in ischemic muscle, hypoxic C2C12 cells, and circDB-knockdown cells (n = 6). (E) TargetScan prediction of miR-34a binding sites in the 3′ UTR of USP7 mRNA. (F) Western blot analysis of USP7 and Notch1 protein levels in C2C12 cells transfected with miR-34a mimic or inhibitor (n = 4). Data are shown as mean ± SEM.

Article Snippet: Membranes were incubated with primary antibodies against USP7 (#sc-376912), Notch1 (#sc-376403), CD9 (#sc-13118), GAPDH (#sc-32233; all from Santa Cruz Biotechnology), and K48-Ub (#8081; Cell Signaling Technology).

Techniques: Expressing, Binding Assay, Knockdown, Western Blot, Transfection

Journal: Regenerative Therapy

Article Title: Extracellular vesicles-delivered circDB promotes ischemic muscle repair through the miR-34a/USP7/Notch1 signaling pathway

doi: 10.1016/j.reth.2025.08.009

Figure Lengend Snippet: Proposed model of the circDB/miR-34a/USP7/Notch1 regulatory axis in ischemic repair . UMSC-EVs deliver circDB to ischemic muscle tissue, where it functions as a sponge for miR-34a. This suppression of miR-34a enhances USP7 expression. USP7, in turn, deubiquitinates and stabilizes Notch1, promoting myoblast proliferation and tissue repair following hindlimb ischemia.

Article Snippet: Membranes were incubated with primary antibodies against USP7 (#sc-376912), Notch1 (#sc-376403), CD9 (#sc-13118), GAPDH (#sc-32233; all from Santa Cruz Biotechnology), and K48-Ub (#8081; Cell Signaling Technology).

Techniques: Expressing