Fig. 4.

R406 promotes the interaction of TAK1 with ubiquitinated RIPK1. (A) Tak1^−/− MEFs were retrovirally reconstituted with the Flag-tagged TAK1 (Flag-TAK1) and the catalytic dead mutant Flag-TAK1(K63W). The cells were subsequently pretreated with or without 3 μM R406 for 30 min and then stimulated with 20 ng ml⁻¹ TNFα for indicated time points. The cells were lysed with 0.5% Nonidet P-40 buffer and cell lysates were immunoprecipitated with anti-Flag antibody-conjugated agarose. All immunoprecipitated complexes, and whole-cell lysates were analyzed by western blotting with the indicated antibodies. (B) MEFs were pretreated with or without 3 μM R406 for 30 min, and then 20 ng/mL TNFα was added for indicated time points. The cells were lysed with 0.5% Nonidet P-40 buffer and cell lysates were immunoprecipitated with anti-TAK1 antibody. All immunoprecipitated complexes and whole-cell lysates were analyzed by western blotting with the indicated antibodies. (C) Flag-RIPK1 MEFs were treated with or without 3 μM R406 for 30 min, and then 20 ng/mL TNFα was added for 30 min, DMSO as control. The cell lysates were immunoprecipitated with anti-Flag beads, the RIPK1 immunocomplexes were eluted with glycine followed by trypsin digestion, phosphor-peptides were enriched and analyzed by mass spectrometry.