Figure 2. DMF-dependent modification of Trx leads to inhibition of NFκB activity.

(A) Cells were treated with DMF (50 μM) in a time-dependent manner as indicated. CEM cells were incubated with H₂O₂ (100 μM) for 2 h as positive control. Reduced proteins were pulled-down using biotinylated iodoacetamide (BIAM) and probed for phospho- and total NFκB. (B) CEM cells were left untreated or treated with DMF (50 μM) for 4 h. NFκB DNA binding was assessed by oligo pull-down (o.p.d.) for indicated biotinylated oligonucleotides containing NFκB binding sites. Positive and negative controls are denoted by P or N respectively (left). Band intensities were quantified from 3 independent experiments and normalized to negative control (right). (C) Quantitative real-time PCR of indicated genes was performed in triplicates using RNA derived from cells treated with DMF (50 μM) for 2, 4, and 8 h. (D) HH, SeAx and CEM cells were incubated with DMF (50 μM) for the indicated time points. Cell lysastes were analyzed by Western blot. (B,C) Error bars represent standard deviation.