PP2Ac-siRNA increases AP1 activity in SLE T cells by increasing the binding of pCREB to the c-fos promoter. (A and B) PP2Ac-siRNA increases AP1 activity in SLE T cells. An AP1 binding [³²P]-radiolabeled oligonucleotide was used in EMSA, where AP1 binding activity was assessed in nuclear extracts derived from cells that were transfected in the presence or absence of PP2Ac-siRNA and subsequently stimulated for 2 hours. Results from a representative experiment with 1 SLE patient and 1 normal subject are shown (A). (B) Mean ± SEM of AP1 binding from 3 patients with SLE and 3 normal subjects examined in parallel are displayed. AP1 binding was quantitated by densitometry of autoradiographed bands. P values are also shown. (C and D) PP2Ac-siRNA–mediated pCREB upregulation parallels increased binding of pCREB to the c-fos promoter and increased binding of both pCREB and c-fos to the IL2 promoter. Normal T cells were transfected with either PP2Ac- or control siRNA and then stimulated with PMA/A23187 for 1–3 hours. pCREB expression was assessed with Western blots (C), and in vivo DNA binding of pCREB and c-fos was examined using ChIP assays (D) as described in Methods. Anti-E47 immunoprecipitates were used as negative control. Results from a representative of 3 similar experiments are shown. (E) T cells were transfected with wild-type PP2Ac or H118N and then stimulated with PMA/A23187 for 1 hour. DNA binding of pCREB and c-fos to the IL2 promoter was examined with ChIP assays (OD values corresponding to each sample are noted below the panel). Results from 1 of 2 similar experiments is shown.