Fig. 4: Caspase-3 cleaves RelA at D97, RelB at D205 and c-Rel at D86.
From: Apoptotic caspase inhibits innate immune signaling by cleaving NF-κBs in both Mammals and Flies
A Immunoblot of purified N-terminal GST and C-terminal Flag tagged proteins as indicated incubated without (−) or with caspase-3 (+) for 60 min at 37 °C. The blot was probed with anti-Flag antibody. B Immunoblot of purified WT and D97A mutant of RelA proteins incubated without (−) or with caspase-3 (+) for 60 min at 37 °C. The blot was probed with anti-Flag antibody. C Quantitative RT-PCR analysis of CXCL10 mRNA in HEK293T cells transfected with RelA and its mutants or truncations followed by immunoblot analysis. D Quantitative RT-PCR analysis of CXCL10 mRNA in HEK293T cells transfected with RelA (p65), its mutant and caspase-3 as indicated. E 293T cells were transfected with Flag-p65 and HA-tagged protease-defective mutant CASP3. Co-immunoprecipitations were performed by using anti-HA or anti-Flag antibody, followed by immunoblot analysis with indicated antibodies. F Alignment of the amino acid sequence of RelA, RelB and c-Rel, the putative caspase-3 recognition motif DXXD was highlighted (red). G Immunoblot of purified WT and D205A mutant of RelB proteins incubated without (−) or with caspase-3 (+). The blot was probed with anti-Flag antibody. H, I Quantitative RT-PCR analysis of CXCL10 mRNA in HEK293T cells transfected with RelB, its mutants or truncations (H) and caspase-3 (I) as indicated. J Immunoblot of purified WT, D86A and D205A mutant of c-Rel proteins incubated without (−) or with caspase-3 (+). The blot was probed with anti-Flag antibody. K, L Quantitative RT-PCR analysis of CXCL10 mRNA in HEK293T cells transfected with c-Rel, its mutants or truncations (K) and caspase-3 (L) as indicated. Data are representative of three independent experiments (means with SEMs). *p < 0.05, **p < 0.01, and ***p < 0.001.
