There is stimulus-dependent phosphorylation of IκB in normal but not patient T cells. T cells isolated from a healthy donor were stimulated with PMA (20 ng/mL) and ionomycin (0.75 μg/mL) and subjected to cytoplasmic extract preparation. (A) Immunoblotting of the cytoplasmic extracts shows the presence of 2 immunoreactive bands within 30 minutes of stimulation. (B) The antibody anti-IκB was incubated with different concentrations of IκB peptide before incubation with membrane and immunoblotting. (C) The cytoplasmic extracts with and without phosphatase inhibitors were incubated with buffer containing 24 U of CIP (+) or buffer alone (−). Equal amounts of the cytoplasmic extract were analyzed by immunoblotting with anti-IκB antibody. The phosphorylated band of IκB is indicated by P-IκB. The upper band of IκB was not detectable in any of the cell lysates from the 7 RCC patients studied. These findings suggest that phosphorylation of IκB was inhibited in T cells from RCC patients.