FIGURE 5.

JMJD5 hydroxylated NFATc1 protein through its enzymatic activity. A, diagram of the full length and three generated fragments (NFATc1-N, -M, and -C) of the mNFATc1 protein. B and C, GST-fused proteins were expressed in E. coli and purified by a two-step purification, glutathione-Sepharose and then ion exchange chromatography (Mono Q column). Purified GST-JMJD5 and the indicated GST-fused substrate proteins were mixed and incubated together with ¹⁴C-radiolabeled 2-OG. 2-OG decarboxylation was measured by ¹⁴CO₂ formation. Values are expressed as relative -fold induction compared with GST only. The error bars represent S.D. values from at least triplicate experiments. The p value for statistical difference was calculated using a two-tailed Student's t test and is indicated by asterisks. GST-fused proteins were visualized by Coomassie Brilliant Blue (CBB) staining. a.a, amino acids. Cn, calcineurin; RHD, Rel homology domain; IPT, immunoglobulin-like fold, Plexins, transcription factors.