Fig. 3

Proteins from resting and stimulated T cells differentially interact with several cis-acting elements. (A) Electrophoretic mobility shift assays were carried out with AP-1, NFκB, NFAT, or RATS probes corresponding to elements of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) and the PRRE (PU) of the interleukin-2 (IL-2) promoter. Proteins employed were from unstimulated (T0) and 4 hr-stimulated (T4) peripheral blood (PB) lymphocytes or from cells stimulated for 4 hr in the presence of the immunosuppressive drug cyclosporin A (T4^CsA). In the upper right panel, antibodies directed against the NFκB components p50 and p65, respectively, produced the supershift indicated by the arrowhead. The NFAT, RATS, and PU bandshifts form a fast-migrating complex with proteins from resting cells, corresponding to the repressor (R), and a slow-migrating complex due to the activator (A). Proteins from unstimulated and 4 hr-activated Jurkat cells form complexes of different size (NFAT). Φ, no protein added. (B) Competition experiments were carried out with labeled probe corresponding to the IL-2 PRRE (PU) or the HIV-1 RATS element and extract displaying repressor activity from resting unstimulated PB lymphocytes (T0 in A). Cold oligonucleotides were added in the excess indicated on the bottom.