anti nicd antibody  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Caveolin-1 modulates Notch transcriptional activity during in vitro respiratory multiciliated cell maturation

doi: 10.1038/s41598-026-40201-6

Figure Lengend Snippet: Analysis of Notch1 Expression and Subcellular Distribution in Luc-KD and Cav1-KD Cells. ( a ) Maximal projection of confocal images for Notch1 (in yellow), and nucleus (in blue) in Luc-KD and Cav1-KD cells in ALI 6. ( b ) Western blot images of Luc-KD and Cav1-KD cells showing Notch 1 (full-length at 300 kDa and cleaved at 120 kDa) and E-Cadherin expression. The lower Western blot image of Notch1 (300 kDa) in the panel shows a contrast-enhanced version of Notch1 region of interest (ROI) amplified to the entire image, derived from the original image with lower contrast shown in the upper part of the panel. Each condition was tested in triplicate (L1, L2, and L3 for each genotype). Cell lysates (L) were derived from an independent cell culture. β-actin was used as a loading control. ( c – e ) Relative protein expression levels quantification of Notch1 300 kDa ( c ), Notch1 120 kDa ( d ) and E-Cadherin ( e ). Mean and standard deviation as error bars were plotted, n = 3 independent lysates per group. ( f ) Analyses of Notch 1 full-length and processed forms (TMD+NICD and NICD) subcellular distribution in the cytosol (Cyt.), membrane (Mem.), and chromatin (Chr.) in Luc-KD and Cav1-KD cells. Cell fractionation and gradient SDS-Gels were used for improved resolution. ( g ) Relative protein expression quantification of Notch 1 subcellular distribution in Luc-KD and Cav1-KD cells. Mean and standard deviation as error bars were plotted, n = 4 independent lysates per group. ( h ) Proposed working model of the mechanism by which Cav-1 regulates BSC differentiation, involving differential NICD binding capacity to chromatin together with other partners. Scale bar in panel a represent 20 μm. p-values in all conditions were obtained using two-tailed t-test (*** represents p < 0.001, ** represents p < 0.01 and n.s. means no significative differences).

Article Snippet: The remaining chromatin was diluted, precleared with protein A/G Sepharose beads, and immunoprecipitated overnight at 4 °C using an anti-NICD antibody (Cell Signaling, #4147S, 2.5 μL of antibody/10 μg of chromatin).

Techniques: Expressing, Western Blot, Amplification, Derivative Assay, Cell Culture, Control, Standard Deviation, Membrane, Cell Fractionation, Binding Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Caveolin-1 modulates Notch transcriptional activity during in vitro respiratory multiciliated cell maturation

doi: 10.1038/s41598-026-40201-6

Figure Lengend Snippet: Analysis of Notch2 Expression and Subcellular Distribution in Luc-KD and Cav1-KD Cells. ( a ) Maximal projection of confocal images for Notch2 (in green), and nucleus (in blue) in Luc-KD and Cav1-KD cells in ALI 6. ( b ) Western blot images of Luc-KD and Cav1-KD cells showing Notch 2 (full-length at 300 kDa and cleaved at 110 kDa) and c-Myb expression. Each condition was tested in triplicate (L1, L2, and L3 for each genotype). Cell lysates (L) were derived from an independent cell culture. GAPDH was used as a loading control. ( c – e ) Relative protein expression levels quantification of Notch2 300 kDa ( c ), Notch2 120 kDa ( d ) and c-Myb ( e ). Mean and standard deviation as error bars were plotted, n = 3 independent lysates per group. ( f ) Analyses of Notch 2 full-length and processed forms (TMD+NICD and NICD) subcellular distribution in the cytosol (Cyt.), membrane (Mem.), and chromatin (Chr.) in Luc-KD and Cav1-KD cells. Cell fractionation and gradient SDS-Gels were used for improved resolution. ( g ) Relative protein expression quantification of Notch 2 subcellular distribution in Luc-KD and Cav1-KD cells. Mean and standard deviation as error bars were plotted, n = 4 independent lysates per group. Scale bar in panel a represent 20 μm. p-values in all conditions were obtained using two-tailed t-test (**represents p < 0.01, *represents p < 0.05 and n.s. means no significative differences).

Article Snippet: The remaining chromatin was diluted, precleared with protein A/G Sepharose beads, and immunoprecipitated overnight at 4 °C using an anti-NICD antibody (Cell Signaling, #4147S, 2.5 μL of antibody/10 μg of chromatin).

Techniques: Expressing, Western Blot, Derivative Assay, Cell Culture, Control, Standard Deviation, Membrane, Cell Fractionation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Alpha hemolysin enhances the immune response by modulating dendritic cell differentiation via ADAM10-Notch signaling

doi: 10.1038/s41392-025-02432-3

Figure Lengend Snippet: Hla H35A activates Notch signaling via ADAM10 in Fl-BMDCs. a Representative western blot (left) and quantification (right) of N1ICD, N2ICD, Notch3, or Notch4 in Fl-BMDCs after 4.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). b Quantification of Hey1 , Hes1 , or Hes5 mRNA expression by qPCR in Fl-BMDCs after 6 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). c Quantification of Adam10 , Notch1 , Notch2 , Notch3 , or Notch4 mRNA expression by qPCR in Fl-BMDCs after 4.5 h of treatment with PBS, PA0833, HPF, or HPF with GI254023X ( n = 3 per group). d TPMs of the Notch1 , Notch2 , Notch3 , or Notch4 genes in unstimulated Fl-BMDCs from bulk RNA-seq ( n = 3 per group) . e Quantification of Notch1 , Notch2 , Notch3 , or Notch4 mRNA expression by qPCR in Fl-BMDCs under homeostatic conditions ( n = 3 per group). Each data point indicates a biological replicate in ( a ) and ( d ). Each data point indicates a biological replicate derived from the average of three technical replicates in ( b , c and e ). The data are presented as the means ± s.e.m.s. Statistical significance was tested via one-way ANOVA followed by Tukey’s multiple comparisons test in ( a – e )

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST (0.1% Tween-20 in TBS) for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against N1ICD (1:1000, CST, Cat# 8216S), N2ICD (1:1000, CST, Cat# 4970T), β-actin (1:1000, CST, Cat# 5732T), Notch3 (1:1000, Santa Cruz, Cat# sc-515825), or Notch4 (1:1000, Santa Cruz, Cat# sc-393893).

Techniques: Western Blot, Expressing, RNA Sequencing, Derivative Assay

Journal: Nagoya Journal of Medical Science

Article Title: XXYLT1 inhibits NOTCH1 activation in Jurkat cells while promoting cell proliferation

doi: 10.18999/nagjms.87.3.431

Figure Lengend Snippet: NOTCH1 EGF repeats overexpressed in Jurkat cells are modified with the O- glucose trisaccharide at EGF10 Fig. 1A: HCD-MS/MS spectra of a glycopeptide from NOTCH1EGF10 carrying the O- Glc trisaccharide Xyl-Xyl-Glc. Numerous b and y ions in addition to the sequential neutral loss of sugar residues from the precursor ion confirm the identity of the peptide and the presence of the O- Glc trisaccharide. Fig. 1B–E: EICs of glycopeptides from EGF10 modified with previously reported glycoforms. Blue circles, glucose; Orange stars, Xyl; White circles, uncharacterized hexose; Black dashed line, naked peptide; Blue, peptide + O- Glc; Yellow, peptide + O- Glc-Xyl; Orange, peptide + O- Glc-Xyl-Xyl; Gray, peptide + 2 hexoses; Purple, peptide + 2 hexoses and sialic acid. (B) WT Jurkat cells, (C) Cas9 control cells, (D) XXYLT1 KO #1 (gRNA-3 transfectant), and (E) XXYLT1 KO #3 (gRNA-1 transfectant). EGF: epidermal growth factor-like HCD: higher-energy collision dissociation MS/MS: tandem mass spectrometry Glc: glucose Xyl: xylose EICs: extracted ion chromatograms WT: wild-type Cas9: CRISPR-associated protein 9 XXYLT1: xyloside α1-3xylosyltransferase 1 KO: knockout gRNA: guide RNA

Article Snippet: Total cell lysates (5 μg) were loaded and subsequently analyzed by western blotting using anti-αTubulin antibody (DSHB, 12G10, 1:4000), anti-cleaved NOTCH1 (Val1744) antibody (CST, D3B8, 1:1000), and anti-HA antibody (MBL, 561, 1:1000), and the intensity of cleaved NOTCH1 was normalized to the intensity of αTubulin.

Techniques: Modification, Tandem Mass Spectroscopy, Glycoproteomics, Control, Transfection, Mass Spectrometry, CRISPR, Knock-Out

Journal: Nagoya Journal of Medical Science

Article Title: XXYLT1 inhibits NOTCH1 activation in Jurkat cells while promoting cell proliferation

doi: 10.18999/nagjms.87.3.431

Figure Lengend Snippet: The activation of NOTCH1 is enhanced in XXYLT1 KO Jurkat clones Fig. 2A: Western Blot analysis of WT, Cas9-control, and XXYLT1 KO clones using antibodies against activated NOTCH1 (Val1744) and α-tubulin (loading control). Fig. 2B: Normalized intensities of activated NOTCH1 signals from five independent experiments. Error bars indicate standard deviation. Significance was calculated using a one-way ANOVA followed by Dunnett’s test. Fig. 2C: Western Blot analysis of NOTCH1 activation in empty vector or XXYLT1-HA -transduced WT and XXYLT1 KO cells. Fig. 2D: Normalized intensities of activated NOTCH1 signals from three independent experiments. Error bars indicate standard deviation. Significance was calculated using a one-way ANOVA followed by Tukey’s test. Fig. 2E: Flow cytometry analysis of cell surface NOTCH1 in WT, Cas9-control, and XXYLT1 KO clones. Normalized mean fluorescence intensities from four independent experiments were plotted. Error bars represent the standard deviation. Significance was calculated using a one-way ANOVA followed by Dunnett’s test. XXYLT1: xyloside α1-3xylosyltransferase 1 KO: knockout WT: wild-type Cas9: CRISPR-associated protein 9 ANOVA: analysis of variance HA: hemagglutinin tag MFI: mean fluorescence intensity

Article Snippet: Total cell lysates (5 μg) were loaded and subsequently analyzed by western blotting using anti-αTubulin antibody (DSHB, 12G10, 1:4000), anti-cleaved NOTCH1 (Val1744) antibody (CST, D3B8, 1:1000), and anti-HA antibody (MBL, 561, 1:1000), and the intensity of cleaved NOTCH1 was normalized to the intensity of αTubulin.

Techniques: Activation Assay, Clone Assay, Western Blot, Control, Standard Deviation, Plasmid Preparation, Flow Cytometry, Fluorescence, Knock-Out, CRISPR