FIGURE 6.
Citrullination of IKKγ by PAD2. A, detection of citrullination in IKKγ immunoprecipitates (IP) from RAW 264.7 cells. Cells were incubated with 1 μg/ml LPS for 4 h after pretreatment with BAPTA/AM (30 μm) for 1 h, and immunoprecipitation was performed with an anti-IKKγ antibody (a-IKKγ). The immunoprecipitates were then immunoblotted (IB) with anti-citrulline antibody (a-citrulline). Proteincit, citrullinated proteins. B, in vitro citrullination assay. Purified IKKγ and PAD2 proteins were incubated in reaction buffer containing 10 mm Ca2+ for 2 h. The reaction mixture was then immunoblotted with the anti-citrulline antibody. Silver staining was done with sister mixture loaded on the gel (bottom panel). IKKγcit, citrullinated IKKγ.