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. 2013 Jul 1;110(29):11851–11856. doi: 10.1073/pnas.1308362110

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Fig. 2. — PAD4 targets GSK3β for citrullination in MCF7 cells. (A) Recombinant GSK3β was incubated with recombinant GST-PAD4wt, GST-PAD4mut (inactive), or left untreated in the presence of calcium. Reaction products were analyzed by Western blot using modified-citrulline and GSK3β antibodies. (B) Interaction of PAD4 with GSK3β in MCF7 cells. Nuclear extracts of MCF7 cells stably overexpressing PAD4-FLAG were immunoprecipitated (IP) with antibodies against either FLAG-tag or GSK3β. Complexes were then immunoblotted (IB) using antibodies against the indicated proteins. (C) Citrullination of GSK3β by PAD4 in MCF7 cells. After treatment with calcium ionophore A23187, nuclear extracts of MCF7 cells overexpressing either PAD4wt-FLAG or PAD4mut-FLAG were immunoprecipitated (IP) with an antibody against GSK3β. Immunoprecipitation material was then immunoblotted with antibodies against GSK3β and modified citrulline.