NF-κB regulates expression of a distinct set of antiapoptotic genes in H/RS cells.

(A) L428 cells were infected with Ad5-control or Ad5-IκBΔN, respectively, and total RNA was prepared 24 hours (L428c24, L428i24) or 48 hours (L428c48, L428i48) after infection. Additionally, RNA was prepared from different control and H/RS cells. The ribonuclease protection assay was performed according to the supplier's instructions (BD Pharmingen). Human apoptosis template set hAPO-5 was labeled with α-³²P UTP. A total of 10 μg RNA and 8 × 10⁵ cpm of labeled probes were used for hybridization. (B) Ribonuclease protection assay using human apoptosis template set hAPO-2b was performed as described in panel A. A total of 10 μg RNA and 3 × 10⁶ cpm of labeled probes were used for hybridization. (C) Whole-cell extracts of different control and H/RS cells or infected L428 cells (as indicated) were analyzed by Western blotting with anti-TRAF1 (upper panel) or anti–c-IAP2 (lower panel) antibodies. (D) Whole-cell extracts, as in panel C, were analyzed in Western blots for Bcl-x_L (upper panel) or Bfl-1/A1 (lower panel). (E) Expression of p53. Whole-cell extracts of noninfected and infected L428 cells were prepared at the indicated time points and analyzed by Western blotting with anti-p53 antibody. NI indicates noninfected.