Figure 7.

Characterization of p65–R274A–S276A (RAPA) knock-in mice. (A) p50 (lane 1), wild-type p65 (lane 2), p50 and wild-type p65 (lane 3), or p50 and RAPA p65 mutant (lane 4) were transiently transfected into rela^−/− 3T3 cells. EMSA using labeled κB probe was performed with nuclear extracts of transfected cells. (B) Sequencing of p65 cDNA obtained by RT–PCR from wild-type and RAPA homozygous mice. (C) Genotype analysis of offspring derived from RAPA heterozygous parents. (D) Wild-type and RAPA/RAPA embryos from 15.5 dpc are shown. The RAPA embryos develop normally and are in general of similar size as the wild-type embryos. (E) Haematoxylin and eosin staining for sections of liver from 15.5-dpc wild-type and RAPA/RAPA embryos is shown. The massive apoptosis in the RAPA/RAPA knock-in livers is apparent. (F) p65 translocation in MEFs upon LPS stimulation detected by immunoblotting of nuclear extracts. (G) EMSA using labeled κB probe and control NF-Y probe. (Top panels) Nuclear extracts (5 μg) from unstimulated MEFs and MEFs stimulated with TNFα or LPS were analyzed. (Bottom panels) Degradation and resynthesis of IκBα protein in MEFs following TNFα or LPS stimulation. (H) Supershift analysis of NF-κB–DNA-binding complexes was performed using indicated antibodies. Nuclear extracts (5 μg) from unstimulated MEFs and MEFs stimulated with TNFα were analyzed. In RAPA MEFs, p50:p65–RAPA complex failed to bind to DNA; instead, the major NF-κB complex on DNA is p50:c-Rel. (I) Transactivation by RAPA p65 detected by reporter gene assay in MEFs. MEFs were transfected with pBIIx-luc and control Renilla construct for 24 h and were stimulated with TNFα, IL-1, or LPS for 6 h, and luciferase activity was analyzed. (+/+) Wild-type; (+/m) +/RAPA; (m/m) RAPA/RAPA.