PARP-1 regulates Smad3 phosphorylation and Smad3 binding at the Foxp3 enhancer. (A) Western blot of P-Smad2, Smad2 and GAPDH expressions in freshly isolated CD4⁺CD25⁻ T cells and CD4⁺CD25⁻ T cells stimulated for 15 minutes with CD3 (5 µg/mL) plus CD28 (2 µg/mL) antibodies in the absence or presence of TGF-β1 (2 ng/mL). Data are representative of 3 independent experiments. (B) Summarization of 3× P-Smad2 western blot analysis. Western blot bands were analyzed by Odyssey software, the PARP-1^+/+ P-Smad2 expression was set as 1, PARP-1^−/− P-Smad2 expression was presented relative to PARP-1^+/+. *P < .05, unpaired 1-tailed Student t test. (C) ChIP-coupled quantitative PCR analysis of Smad3 enrichment in the enhancer region of Foxp3 gene assessed using an antibody to Smad3 and presented relative to input and compared with a control IgG. Data are representative of 4 independent experiments (mean ± SEM); ***P < .001 (unpaired 2-tailed Student t test).