2-AA pretreatment modulates activation of the NF-κB pathway in mouse macrophages.

<p>(A) Schematic of 2-AA treatment. Macrophages were left untreated (No Pre) or pretreated with 0.8-mM 2-AA or 4-AA for 48 h (2-AA/4-AA Pre). The untreated and 2-AA pretreated cells were then stimulated with 0.2 mM, 0.4 mM, or 2.0 mM 2-AA (for experiment in B) or 4-AA (for experiment in C). (B) Pretreatment with 2-AA blocked NF-κB activation relative to cells not pretreated with 2-AA (0.8 mM). (C) NF-κB was activated by 2-AA analog 4-AA in 4-AA pretreated and not pretreated cells. Mean values calculated from 2–4 replicate experiments are depicted with SD error bars. (D and E) Following stimulation with 2-AA (0.4 mM), cellular extracts prepared from not pretreated and 2-AA pretreated macrophages. Western blots of I-κBα and I-κBβ degradation (D) and phosphorylation of NF-κB subunit p65 (E). Loading was normalized relative to mouse β-actin. (F and G) A TransAM NF-κB assay showed binding of NF-κB p65 and p50 with the NF-κB promoter in not pretreated and 2-AA pretreated cells following stimulation with 2-AA. Mean values calculated from three replicate experiments are depicted with SD error bars. (p<0.05, Student's t test).