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. 2019 Dec 9;5:151. doi: 10.1038/s41420-019-0230-2

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Fig. 5 — a A predicted BH3-like domain in human MLKL is partially conserved across the indicated species. The consensus sequence for a BH3 domain is shown below. b Human MLKL interacts with Bcl-2. Various combinations of Flag-MLKL, Flag-MLKL L176A, Bcl-2, and Bcl-2 G145A were transfected into HEK-293T cells. Bcl-2 antibodies were used to immunoprecipitate the Bcl-2 proteins. The upper two blot panels show the co-immunoprecipitated Flag-MLKL and the immunoprecipitated Bcl-2 proteins. The lower three panels show the input proteins in cell lysates. c Phosphorylated MLKL interacts better with Bcl-2. Flag-MLKL, Flag-MLKL TS357-58ED (ED), and Flag-MLKL TS357-58AA (AA) were expressed in HEK-293T cells. The wild type and the AA mutant were also co-expressed with RIP3 The ED mutant is a phosphomimetic of RIP3 phosphorylated MLKL while the AA mutant cannot be phosphorylated at TS357-58. Anti-flag antibodies were used for co-immunoprecipitating Bcl-2. Western blots show all indicated proteins expressed in HEK-293T cells. d The endogenous Bcl-2 interacts the endogenous MLKL in THP-1 cells. The PMA treated THP-1 were used for immunoprecipitating Bcl-2 with or without treatment of TNFα 20 ng/ml (T), 100 nM Smac mimetic (S) and 20 µM z-VAD-FMK (Z) for 3 h. The lysates were used d for Bcl-2 immunoprecipitations. The pulled down of MLKL and the input proteins were blotted. (e) Bcl-2 recombinant protein binds with MLKL in vitro. The immunoprecipitated MLKL and its L176A mutant from HEK 293T cells were incubated with Bcl-2 protein for 1 h, and then heavily washed. Blots show the indicated proteins. f GST fusion BH3-like domain of MLKL binds Bcl-2 recombinant protein. The indicated GST fusion BH3 domains were expressed and purified from HEK-293T cells. Bcl-2 recombinant protein was added for in vitro binding with GST fusion BH3 domains.