Figure 3.

MLKL is indeed cleaved upon cell death induction in MM cells. (A) Endogenous MLKL immunoprecipitates prepared from DMSO or bortezomib-treated OPM2 and L363 cells were analyzed by western blotting with antibody for C-terminus. Protein G Dynabeads with lysis buffer was used as negative control and the detected band around 55 kDa was the IgG heavy chain. The input lysates were also analyzed by western blotting. (B) OPM2 and L363 cells were transiently transfected with 5 µg C-terminal Flag-MLKL vector encoding wild-type MLKL. After 18 h, exogenous MLKL was immunoprecipitated from transfected or non-transfected cells with anti-Flag M2 beads. The input lysates and immunoprecipitates were analyzed by western blotting with indicated antibodies. (C) Outline of MLKL cleavage study experiment. Exogenous MLKL immunoprecipitates obtained as described in (B) were subjected to reducing SDS-PAGE and proteins were stained with Coomassie stain. Then, total MLKL and C-terminus bands were excised and performed for mass spectrometric analysis. (D) Identified Peptides from total MLKL and C-terminus by mass spectrometric analysis. M, the specific amino acid was modified by oxidation.