nicd  (Cell Signaling Technology Inc)

Journal: Pancreas

Article Title: Knockdown of Lymphoid Enhancer-binding Factor 1 Inhibits Pancreatic Adenocarcinoma Growth and Neoangiogenesis by Curbing Notch1 and Nuclear Factor Kappa B Signaling Pathways

doi: 10.1097/MPA.0000000000002562

Figure Lengend Snippet: Knockdown of LEF1 interferes with PAAD cell malignant progression and neovascularization by inhibiting Notch1 and NF-κB signaling pathways. A–F, Immunofluorescence experiments and fluorescence intensity analysis: the relative fluorescence level of Notch1 and P65 decreased in AsPC-1/LEF1 and BxPC-3/LEF1. G–M, WB assay results and quantitative analysis: the total Notch1, NICD, Hes1, Hey1, and Jagged1 protein levels and the p-P65/P65 ratio were decreased in AsPC-1/LEF1 and BxPC-3/LEF1 (n = 3, * P <0.05).

Article Snippet: The first antibodies were as follows: LEF1 (ab137872, 1:1000, Abcam), proliferating cell nuclear antigen (PCNA, ab29, 1:1000, Abcam), MMP-2 (4022S, 1:1000, CST), MMP-9 (3852S, 1:1000, CST), BAX (2772S, 1:1000, CST), Bcl-2 (15071S, 1:1000, CST), Cleaved caspase-3 (9661S, 1:1000, CST), Cleaved caspase-9 (9509S, 1:1000, CST), vascular endothelial growth factor-A (VEGFA, ab214424, 1:1000, Abcam), total Notch1 (ab52627, 1:1000, Abcam), NICD (4147, 1:1000, CST), Hes1 (ab71559, 1:1000, Abcam), Hey1 (ab235173, 1:1000, Abcam), Jagged1 (ab300561, 1:1000, Abcam), P65 (ab32536, 1:1000, Abcam), p-P65 (3033S, 1:1000, CST), GAPDH (5174S, 1:5000, CST), HRP-anti-Rabbit (A0362, 1:1000, Beyotime), HRP-anti-Mouse (A0350, 1:1000, Beyotime).

Techniques: Knockdown, Protein-Protein interactions, Immunofluorescence, Fluorescence

Journal: Pancreas

Article Title: Knockdown of Lymphoid Enhancer-binding Factor 1 Inhibits Pancreatic Adenocarcinoma Growth and Neoangiogenesis by Curbing Notch1 and Nuclear Factor Kappa B Signaling Pathways

doi: 10.1097/MPA.0000000000002562

Figure Lengend Snippet: Knockdown of LEF1 enhances immune response in mice. A and B, Immunofluorescence results and percentage of positive cells: the fluorescence intensity of PD-L1 in tumor tissues of sh-LEF1 group decreased. C and D, HE staining results and necrotic cell count showed that the proportion of cell necrosis in sh-LEF1 tumor tissue was decreased. E–H, ELISA results and content statistics showed that knockdown of LEF1 downregulated the contents of TGF-β1, IL-10, PD-L1, and CCL2 in sh-LEF1 tumor tissues. I–O, WB assay results and quantitative analysis showed decreased levels of total Notch1, NICD, Hes1, Hey1, and Jagged1 proteins in tumor tissues, and a decreased p-P65/P65 ratio (n = 6, * P <0.05).

Article Snippet: The first antibodies were as follows: LEF1 (ab137872, 1:1000, Abcam), proliferating cell nuclear antigen (PCNA, ab29, 1:1000, Abcam), MMP-2 (4022S, 1:1000, CST), MMP-9 (3852S, 1:1000, CST), BAX (2772S, 1:1000, CST), Bcl-2 (15071S, 1:1000, CST), Cleaved caspase-3 (9661S, 1:1000, CST), Cleaved caspase-9 (9509S, 1:1000, CST), vascular endothelial growth factor-A (VEGFA, ab214424, 1:1000, Abcam), total Notch1 (ab52627, 1:1000, Abcam), NICD (4147, 1:1000, CST), Hes1 (ab71559, 1:1000, Abcam), Hey1 (ab235173, 1:1000, Abcam), Jagged1 (ab300561, 1:1000, Abcam), P65 (ab32536, 1:1000, Abcam), p-P65 (3033S, 1:1000, CST), GAPDH (5174S, 1:5000, CST), HRP-anti-Rabbit (A0362, 1:1000, Beyotime), HRP-anti-Mouse (A0350, 1:1000, Beyotime).

Techniques: Knockdown, Immunofluorescence, Fluorescence, Staining, Cell Characterization, Enzyme-linked Immunosorbent Assay

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