Figure 5.

ROS-signaling regulates Irf8 nuclear accumulation in Alox15 cells. (A) Alox15 BMM have decreased expression of Nf1 and similar levels of Irf8 compared to B6 by real time analysis normalized to Gapdh (n=3 independent experiments). (B–C) Total and nuclear lysates were isolated from B6 and Alox15 BMM and immunoblotted for Irf8 demonstrating (B) Alox15 exhibit comparable expression of total Irf8 protein but (C) decreased expression of nuclear Irf8 protein (n=3 independent experiments.) (D) Lipid product formation in B6 and Alox15 BMM after stimulation with 50 μM AA demonstrating decreased 12(S)HETE and increased 5(S)HETE in Alox15 BMM compared to B6. (n=4 in two independent experiments). (E) Flow cytometric analysis of B6 and Alox15 BMM loaded with the ROS sensitive dye H₂DCFDA demonstrate decreased levels of ROS in Alox15 BMM. Shown are a representative experiment and a summary of 5 experiments. (F) Addition of 10 mM Tiron, a ROS scavenger, to B6 BMM overnight decreases Nf1 transcription while addition of 50 μM BSO, which increases ROS, to Alox15 BMM increases Nf1 transcription measured by real time analysis. n=3 independent experiments. (G) Modulation of ROS regulates Irf8 nuclear accumulation. Nuclear lysates were isolated from B6 BMM in the presence and absence of 10 mM Tiron and Alox15 BMM cultured alone or in the presence of 50 μM and 100 μM BSO overnight. Protein expression of IRF-8 was determined by immunoblot. Shown is an immunoblot from a representative experiment in which an irrelevant lane was removed and a summary of three experiments. *p<.05, **p<.01. Error bars represent ± SEM.