Fig. 6

TRAF6 is essential for the Notch1-mediated inflammatory response and ROS generation in LPS-stimulated macrophages. BMMs were isolated from the Notch1^FL/FL and Notch1^M−KO mice and transfected with CRISPR/Cas9-mediated TRAF6 activation or TRAF6 KO vector. A Western blot analysis of TRAF6, p-TAK1 and p-P65 in BMMs transfected with CRISPR/Cas9-mediated TRAF6 activation and control. Representative of three experiments. B Quantitative RT-PCR analysis of TNF-α, IL-1β, CCL-2 and CXCL-10 mRNA levels in vitro (n = 3–4 samples/group). C Detection of ROS production by Carboxy-H2DFFDA in LPS-stimulated macrophages from the Notch1^FL/FL mice. Quantification of ROS-producing Macrophages (green) (n = 4–6 mice/group). Scale bars, 40 μm. D Western blot analysis of TRAF6, p-TAK1 and p-P65 in BMMs transfected with CRISPR/Cas9-mediated TRAF6 KO and control. Representative of three experiments. E Quantitative RT-PCR analysis of TNF-α, IL-1β, CCL-2 and CXCL-10 mRNA levels in vitro (n = 3–4 samples/group). F Detection of ROS production by Carboxy-H2DFFDA in LPS-stimulated macrophages from the Notch1^M−KO mice. Quantification of ROS-producing Macrophages (green) (n = 4–6 mice/group). Scale bars, 40 μm. All data represent the mean ± SD. *p < 0.05. **p < 0.01. ***p < 0.001