Fig 1: Effect of NOTCH4 expression on NOTCH receptor processing and signaling. (A) (Upper panel) Western blot analysis of furin and ADAM10 expression; (lower panel) furin mRNA analysis by RT-PCR in Raw 264.7 cells transiently transfected with a Notch4 expression vector or the corresponding empty vector, activated with LPS (100 ng/ml) and/or IFN-γ (10 U/ml) for different times. β-tubulin expression was used as a loading reference in Western blots. The image is representative of three independent experiments. RNA expression was referred to that of the P0 housekeeping genes used as a control. Quantitation of three different experiments is shown. One-way ANOVA/Bonferroni’s post-tests were performed. *p<0.05 compared to the corresponding control treated cells. (B) Analysis by Western blot (upper panel) and qRT-PCR (lower panel) of Notch1 gene expression in Raw 264.7 cells transiently transfected with a Notch4 expression vector or the corresponding empty vector, activated with LPS and IFN-γ for different times. β-tubulin expression was used as a loading reference in Western blot. The image is representative of three independent experiments. RNA expression was referred to that of the P0 gene. Quantitation of three different experiments is shown. One-way ANOVA/Bonferroni’s post-tests were performed. *p<0.05 compared to the corresponding control treated cells. (C) Effect of NOTCH4 expression on NOTCH1 transcriptional activity. Raw 264.7 cells were transfected with a CBF-1 luciferase reporter plasmid in the presence of a Notch4 expression vector or the corresponding empty vector, with a full-length Notch1 (Notch1) or Notch1 intracellular domain (NIC1) expression vectors. Cells were activated with LPS for 24 h. The means ± SD of three independent experiments is shown. One-way ANOVA/Bonferroni’s post-tests were performed. ***p<0.001 compared to the corresponding LPS activated, Notch4 untransfected cells, ###p<0.001 compared to the corresponding LPS activated, non transfected with Notch1 expression vectors cells.
Fig 2: DLL4 mRNA expression is upregulated in gastric cancer and DLL4 protein is over-expressed in human GPL specimens. Representative IHC images demonstrating the expression of DLL4 (A), Notch1 (B) and Notch4 (C) in GPL and normal tissues (×200). Semi-quantitative analysis of DLL4 (D), Notch1 (E) and Notch4 (F) protein expression in human specimens (n = 102). # p < 0.05 versus the control group. Data are presented as mean ± SEM. Abbreviations: AT-III, Atractylenolide III; GPL, gastric precancerous lesions; GC, gastric cancer; IHC, immunohistochemistry; SEM, standard error of mean.
Fig 3: Schematic representation of NOTCH4 receptor interference with TLR4 and IFNγ receptor signaling in proinflammatory activated macrophages. NOTCH4 diminishes STAT1 phosphorylation after IFN-γ receptor activation. This lower STAT1 activation decreases the expression of STAT1-dependent genes, including IRF1, but increases the expression of HES1, which is normally repressed by STAT1 (39). HES1 exerts an anti-inflammatory action by inhibiting the expression of genes such as those for IL-6 or IL-12 (8). Moreover, enhanced levels of HES1 could facilitate STAT3 phosphorylation and activation, increasing the expression of anti-inflammatory genes, such as that for IL-10 (7). NOTCH4 also inhibits NF-κB-dependent transcription. This effect is mediated, at least in part, by the lower activation of STAT1, that normally cooperates with NF-κB increasing its transcriptional activity in some pro-inflammatory gene promoters, such as those of IL-6 or IL-12 (36). Because of the lower STAT1 and NF-κB activation mediated by NOTCH4, and of the elevated levels of the repressor HES1, the expression of STAT1 and NF-κB target genes, such as IL-6, IL-12 or CD80 among others, is diminished. The blue arrows mark increased phosphorylation or expression induced by NOTCH4, whereas red arrows indicate repression by NOTCH4.
Fig 4: NOTCH4 expression in myocardial tissue. (A) Immunohistochemical staining for NOTCH4 expression in ROVT myocardial tissue from patients with TOF (n=24) and healthy controls (n=5). NOTCH4 was expressed in both the nucleus and cytoplasm. The staining intensity of NOTCH4 was relatively weaker in patients with TOF. (B) Quantification of immunohistochemical staining. **P<0.01; Mann-Whitney test. TOF, tetralogy of Fallot; ROVT, right ventricular outflow tract.
Fig 5: Aberrant expression of Notch4, Dll4, NF-κB, VEGF, Flt-1 and Flk-1 in the rat lung after intrauterine infection. (A) The mRNA expression levels of Notch4, (B) Dll4, (C) NF-κB, (D) VEGF, (E) Flk-1 and (F) Flt-1 were evaluated in the two groups by reverse transcription-quantitative PCR. (G) The protein expression levels of Notch4, Dll4 and NF-κB, and (H) VEGF, Flt-1 and Flk-1 were determined in the two groups at P3 by western blotting. Data are presented as the mean ± SD (n=10 per group). *P<0.05. Dll4, delta-like canonical Notch ligand 4; Flt-1, FMS-like tyrosine kinase 1; Flk-1, fetal liver kinase 1; P, postnatal day.
Supplier Page from Abcam for Anti-NOTCH4 antibody [EPR18049]