NFκB signalling is affected by TSA. (A) Effect of TSA on levels of nuclear NFκB. CD4+ T-cells were cultured with various concentrations of TSA (+TSA) or DMSO alone (-TSA) for 8 hours, whilst concomitantly stimulated with PMA+ionomycin (50 ng/ml and 1 μg/ml, respectively), or not stimulated (-PMA/ionomycin). Nuclear extracts were analysed for the presence of NFkB by western blot analysis using an antibody raised against NFκB p65 (C-20) (Santa Cruz Biotechnology, Inc. USA). (B) Effect of TSA on IκBα expression. CD4+ T-cells were pretreated with either 100 nM TSA (+TSA) or DMSO alone (-TSA) for 4 hours, whereafter phorbol-12-myristate-13-acetate (PMA; 50 ng/ml) and ionomycin (1 μg/ml), or vehicle (-PMA/ionomycin) was added to the medium and the cells were cultured for various periods of time. Cell lysates were analysed for IκBα expression by Western blot analysis using an anti-IκBα antibody from Cell Signaling Technology (CST, Beverly, MA). Reversible staining of blots with Ponceau S was used as loading and blotting control.