Fig. 3: TRIM28 interacts with MLKL and is phosphorylated at Ser473 in complex IIb during necroptosis.
A, B HT-29 cells were treated with the compounds as indicated. The levels of specific proteins were analyzed by western blotting using indicated antibodies. C, D RIPK1−/− (C) and MLKL−/− (D) MEFs were treated with TSZ as indicated. The levels of total TRIM28 and phosphorylation of TRIM28 S473 were analyzed by western blotting. E HT-29 cells were treated with TSZ or TSZ + Nec-1s for 4 h. Lysates were immunoprecipitated with MLKL antibody and analyzed by western blotting using indicated antibodies. F MEFs were treated with TSZ for 3 h. The cell lysates were immunoprecipitated by pRIPK1S166 antibody. Both lysate input and immunoprecipitation samples were analyzed by western blotting using indicated antibodies. *, non-specific bands. G 293T cells were transfected with GFP-MLKL and HA-TRIM28 for 24 h. The cell lysates were immunoprecipitated with anti-HA resin and analyzed by western blotting using indicated antibodies. H 293T cells were transfected with tet-inducible flag-tagged MLKL Q356A expression vector. MLKL oligomerization was induced by doxycycline with or without NSA. Cell viability was determined by CellTiter-Glo (left). Cell lysates were separated by non-reducing SDS/PAGE and analyzed by western blotting using MLKL antibody (right). I 293T cells were transfected with tet-inducible flag-tagged MLKL Q356A expression vector. After doxycycline induction, the cell lysates were prepared and immunoprecipitated by anti-Flag resin and analyzed by western blotting using indicating antibodies. TNFα, 20 ng/mL; zVAD, 25 μM; SM164, 50 nM; Nec-1s, 10 μM; GSK’872, 10 μM; NSA, 2 μM; Dox, 2 ng/mL. Data were presented as mean ± SEM. ***t-test p < 0.001, n = 3.
