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. 2023 Sep 21;120(39):e2308079120. doi: 10.1073/pnas.2308079120

Fig. 3.

Fig. 3.

R406 promotes the activation of TAK1 kinase. (A) Flag-RIPK1 MEFs were pretreated with or without 3 μM R406 for 30 min and then 20 ng/mL TNFα were added for 5, 15, 30, 60 min. The cells were lysed with 0.5% Nonidet P-40 buffer. The cell lysates were collected and sequentially immunoprecipitated with anti-TNFR1 and anti-Flag antibody. First immunoprecipitation (IP): The TNFR1 complex I was immunoprecipitated using anti-TNFR1 antibody. Second IP: the supernatants after the first IP were then immunoprecipitated with anti-Flag antibody. The 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 were added for 5, 15, 30, 60 min. The cells were lysed with 0.5% Nonidet P-40 buffer. The cell lysates were collected and immunoprecipitated with anti-TNFR1 antibody. The immunoprecipitated complexes and whole-cell lysates were analyzed by western blotting with the indicated antibodies. (C and D) MEFs were treated for 30 min with R406 (3 μM) or 1 h with 5Z-7 (0.5 μM), then stimulated with 20 ng/mL TNFα for indicated time points. The cells were lysed with RIPA buffer. The cell lysates were analyzed by western blotting with the indicated antibodies. (E) MEFs were pretreated with or without R406 (3 μM) and Nec-1s (10 μM) for 30 min, then 20 ng/mL TNFα and 20 μM zVAD.fmk were added for indicated time points. The cells were lysed with 0.5% Nonidet P-40 buffer and cell lysates were immunoprecipitated with anti-RIPK3 antibody. All immunoprecipitated complexes and whole-cell lysates were analyzed by western blotting with the indicated antibodies. (F) MEFs were pretreated with or without 3 μM R406 for 30 min, then 20 ng/mL TNFα and 20 μM. zVAD.fmk were added for indicated time points. The cells were lysed with 0.5% Nonidet P-40 buffer, and cell lysates were immunoprecipitated with anti-FADD antibody. All immunoprecipitated complexes and whole-cell lysates were analyzed by western blotting with the indicated antibodies.