Figure 10. IsoLG adduction of PU.1 disrupts binding to C1q.

Animals were treated with 2-HOBA beginning at 7 weeks of age and sacrificed after 6 weeks of treatment. DCs were harvested and analyzed by RT-PCR. Quantitation of C1qA, C1qB, and C1qC transcripts in (A) B6.SLE123 mice treated with vehicle or 2-HOBA compared with C57BL/6 controls and (B) NZBWF1 mice treated with vehicle or 2-HOBA compared with NZW controls. Data were analyzed with 1-way ANOVA with Tukey’s post hoc test (n = 4–5). (C) Human monocytes were harvested from 6 healthy controls and cultured in the presence of vehicle, GM-CSF, or GM-CSF with a 30-minute exposure to tert-butyl-hydroperoxide (tBHP). Chromatin fragments were incubated with PU.1 antibody on a Sepharose substrate. Precipitated chromatin was eluted, and PCR was performed on extracted DNA. PCR was examined on a 2% agarose gel. (D) Quantitation of band intensity is represented. Data were analyzed with a repeated measures 1-way ANOVA with Tukey’s post hoc test (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Human monocytes were cultured in the presence of GM-CSF with or without a 30-minute exposure to tBHP. Protein lysate was analyzed for PU.1 expression by immunoblot. (F) A 3′ biotin-labeled annealed DNA fragment containing the C1q PU.1 binding site was incubated in the presence of absence of PU.1 or previously isoLG-adducted PU.1. (G) A 3′ biotin-labeled annealed DNA fragment containing the C1q IRF8 binding site was incubated in the presence or absence of IRF8 or previously isoLG-adducted IRF8. Binding reactions were examined on a Tris-borate-EDTA acrylamide gel and imaged on a nylon membrane.