FIG. 6.

Interaction of PKR with IKKβ. (A) Extracts from HeLa cells treated or not treated with 500 U of IFNα per ml were incubated with protein A/G-agarose previously coated with anti-IKKα or anti-IKKβ antibodies. Although PKR specifically interacts with IKKβ (see text), the antibodies directed against IKKβ (Santa Cruz) that we have used are unable to immunoprecipitate IKKβ. Therefore, they were conveniently used here as a control for nonspecific binding (Blank). After extensive washes, the proteins which were retained on the beads were separated by SDS-PAGE on 12.5% acrylamide gels and analyzed by immunoblotting with anti-PKR antibodies. Each lane represents the analysis of proteins immunoprecipitated (IP) from extracts equivalent to 5 × 10⁶ cells. In parallel, crude extracts were analyzed by immunoblotting for the presence of IKKα or PKR. (B) The proteins IKKβ, IKKα, NEMO, PKR/KR296, and luciferase (Luc) were translated in vitro from 2 μg of their respective plasmids in 25 μl of reticulocyte lysate in the presence of ProMix using a TNT-coupled Reticulocyte Lysate System (Promega). The translation products were analyzed by SDS-PAGE either as such (5 μl; 10% input) or after incubation for 18 h at 4°C with 1.25 μg of GST-PKR or an identical amount of GST-HP1α, a nuclear protein (52). Beads were adjusted to 50 μl in all samples with glutathione-Sepharose 4B (Amersham). (C) Cell extracts from 75 × 10⁶ 70Z/3 and 1.3E2 (NEMO-deficient) cells were incubated as described above with GST-PKR or GST-HP1α. Crude extracts corresponding to 7.5 × 10⁶ cells (10% input) and purified extracts were analyzed by immunoblotting with antibodies directed against murine IKKβ after SDS-PAGE.