(A) RCC tumor supernatant suppresses κB-specific binding activity. Human peripheral blood T cells from healthy volunteers were cultured in medium, RCC-S, or NKC-S for 18 hours and then stimulated with PMA (20 ng/mL)/ionomycin (0.75 μg/mL) for 2 hours. Nuclear extracts were isolated and EMSA assays were performed with the oligonucleotide that corresponds to the κB sequence of the IL2R gene. (B) T cells from the healthy donor were cultured with medium or RCC-S for 18 hours and then stimulated with PMA (20 ng/mL)/ionomycin (0.75 μg/mL) for the times indicated. The nuclear and cytoplasmic extracts were prepared and then analyzed by immunoblotting for RelA, c-Rel, NFκB1 (p50), and IκB. Suppression of NFκB activation was observed in 12 of the 16 RCC-S. In addition, 6 of 11 RCC-S tested inhibited IκB phosphorylation and suppressed IκB degradation.