CD28 expression is downregulated by inhibition of Notch signalling. (A) T-ALL cell lines were treated with 10 uM GSI IX (or DMSO) for 24 hrs and cell surface CD28 expression analysed by flow cytometry. * represents p < 0.05 verus DMSO-treated cells. (B) GSI washout experiment as described in figure 2 and CD28 mRNA expression analysis in Jurkat cells. * represents p < 0.05 verus cells at the zero timepoint. ** represents p < 0.05 verus cyclohexamide- cells (C) Real-time PCR expression analysis of CD28 using cDNA from GFP- (untransduced) or GFP+ (transduced) Jurkat cells transduced with GFP-alone or N1ΔE retrovirus. * represents p < 0.05 verus GFP-alone transduced cells. (D) Cell surface CD28 expression using the cells described above. (E) CD28 expression in primary CD3+ cells transduced with GFP-alone, N1ΔE, or N1ΔE+GSIs.