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. 2008 Sep 8;105(37):14023–14027. doi: 10.1073/pnas.0806726105

Fig. 4.

Fig. 4.

IL-6 plus TGF-β down-regulates FOXP3 binding to chromatin that could be reversed by HDACi sodium butyrate. Ten million FOXP3+ SZ-4 T cells were stimulated in the presence or absence of HDACi (10 mM sodium butyrate) along with 20 ng/ml IL-6, 5 ng/ml TGF-β, and 50 U/ml IL-2 as indicated for 4 h in 10% FBS containing RPMI-1640 medium supplied with 10 U/ml IL-2. Cells were washed with cold PBS containing all inhibitors and fractionated into cytoplasmic, nuclear, and chromatin fractions. Equal amounts of proteins were separated by 8% SDS/PAGE, then transferred to nitrocellulose membrane. Both the nuclear fraction (A) and chromatin fraction (B) were immunoblotted successively with antibodies against FOXP3 (221D), Ku-70 (Santa Cruz Biotechnology, sc-17789), and β-actin (Sigma).