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. 2021 Dec 1;12:734966. doi: 10.3389/fimmu.2021.734966

Figure 6.

Figure 6

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.