Fig. 3.

Bay11 blocks phosphorylation of IκBα and NF-κB activity even in the presence of IL-7. Day 4 OT-I CD8 T cells were purified from spleen cells, and 5 × 10⁶ OT-I T cells were cultured with vehicle alone, 10 μM Bay11, 0.6 μg/ml rhIL-7, or IL-7 plus Bay11 for the indicated times. (A) Cell lysates were prepared and immunoblotted with antibodies specific for p-IκBα, IκBα, or β-actin. P+I, Positive control lysates for p-IκBα derived from naïve splenocytes that were stimulated with 125 ng/ml PMA plus 2.5 μg/ml ionomycin for 1 h. β-Actin was used as protein-loading controls. The tabulated values represent the relative ratios of p-IκBα or IκBα to β-actin (relative ratio with β-actin was set at 1.0). (B) A nuclear extract was prepared, and NF-κB DNA-binding activity was examined. Data show p65 and p50 NF-κB activity, and the black bar indicates activity at t = 0 h (before culture), the darker gray bar for the vehicle-treated group, the dark gray for IL-7 treatment, the light gray for Bay11, the lighter gray for IL-7 plus Bay11, and the open bar is a negative control. NF-κB activity data from a 0.5-h culture was performed once, and the experiment for a 2.5-h culture was repeated twice. The summary of data from four other experiments is shown in Table 1. (C) Five million OT-I CD8 T cells were culture for 1 h with 10 μM Bay11 or 50 μM PD98059 and then pulsed or not with 125 ng/ml PMA plus 2.5 μg/ml ionomycin for 10 min. Cell lysates were immunoblotted for p-ERK1/2 (ExpI). The tabulated values show the relative ratios of p-ERK1 to p-ERK2 (relative ratio of p-ERK2 was set as 1.0). In experiment 2 (ExpII), lysates were blotted simultaneously for p-ERK1/2 and p-IκBα and ERK1 and IκBα, the latter two used as protein-loading controls. The tabulated values indicate the relative ratios of p-ERK1 or p-ERK2 to ERK1 and p-IκBα to IκBα.