Fig. 4: AOPPs activated NADPH oxidase and increased intracellular ROS generation by RANK and RAGE signaling.
A BMMs were treated with AOPPs for 0, 15, 30, 60, 120 min, then p47phox were measured by immunofluorescence staining. Representative confocal microscope images of AOPP-induced membrane translocation of p47phox. Scale bars = 30 µm. B Co-immunoprecipitation analysis showed that AOPP treatment enhanced the binding of p47phox to Nox1, Nox4, and p22phox. C Western bolting analysis revealed that AOPPs treatment increased expression of NADPH oxidase subunits Nox1, Nox4, p22phox and p47phox. D Co-immunoprecipitation analysis showed that the enhanced binding of p47phox to Nox1, Nox4, and p22phox induced by AOPPs were inhibited after knockdown of RANK or RAGE. E Intracellular ROS was detected by DCFH-DA. AOPPs treatment induced intracellular ROS generation. F, G AOPPs-induced ROS generation were significantly blocked after knockdown RANK or RAGE or pre-treatment with apocynin (100 μM, a NADPH oxidase inhibitor) or SOD (50 U/ml, a radical scavenger superoxide dismutase). Representative images of three view fields of per experiment were shown. Each co-IP was performed three times experiments and blotted separately each time. Data were presented as mean ± SD. *p < 0.05 versus the vehicle-treated group. #p < 0.05 versus AOPPs-treated group.
