Fig. 7.

Inhibition of Bcl3 expression by BZ is associated with decreased p65 and p50 recruitment, but increased p52 recruitment, to Bcl3 promoter in CTCL cells. (A) Immunoblotting of cytoplasmic (CE) and nuclear (NE) extracts prepared from Hut-78 cells treated 24 h with increasing BZ concentrations, and analyzed by using p65, p50, RelB, cRel and p52 NFκB antibodies. The presence of cytoplasmic proteins in nuclear fraction was evaluated by re-probing the membrane with lactate dehydrogenase (LDH) antibody. Nuclear contamination in the cytoplasmic fraction was assessed by using lamin B specific antibody. To confirm equal protein loading, the membranes were stripped and re-probed with actin antibody. Each lane corresponds to approximately 5 × 10⁴ cells. (B) Schematic illustration of the NFκB binding site in human Bcl3 promoter and primers used in the ChIP assay. (C) Recruitment of NFκB p65, p50, cRel, RelB and p52 subunits to Bcl3 promoter in Hut-78 cells treated 24 h with 0, 10 and 100 nM BZ was analyzed by ChIP and quantified by real time PCR. The data are presented as the change in occupancy over the human IGX1A (SA Biosciences) sequence control and represent the mean ± SE of four experiments. Asterisks denote a statistically significant (p < 0.05) change compared to control untreated cells. (D) Model of the regulation of Bcl3 transcription by the exchange of NFκB subunits in CTCL cells.