Cells were lysed with loading buffer (50 mM Tris pH=6.8, 2%SDS and 10% glycerol) and denatured by boiling. Protein concentration was then determined by the BCA assay (Pierce) and systematically normalized before SDS-PAGE. Following the transfer of proteins to Hybond C nitrocellulose (Amersham Bioscience), immunodetection was performed using the indicated primary and peroxidase-coupled secondary antibodies (Amersham Bioscience). Proteins were visualized by enhanced chemiluminescence (ECL, Amersham Bioscience).
For p-MLKL detection in HT-29 cells, the antibody ab187091 (Abcam) was used. For p-MLKL detection in iMac cells, the antibody ab196436 (Abcam) was used. For p-MLKL detection in NIH3T3 cells, a construct expressing a “humanized” mouse MLKL was retrovirally introduced. In this construct, the human MLKL sequence “ELRKTQTSMSLGTTR” replaced the mouse MLKL sequence “ELSKTQNSISRTAKS”. This “humanized” mouse MLKL is phosphorylated by mouse RIPK3 and is also recognized by human p-MLKL antibody ab187091 (Abcam).
For p-MLKL detection in HT-29 cells, the antibody ab187091 (Abcam) was used. For p-MLKL detection in iMac cells, the antibody ab196436 (Abcam) was used. For p-MLKL detection in NIH3T3 cells, a construct expressing a “humanized” mouse MLKL was retrovirally introduced. In this construct, the human MLKL sequence “ELRKTQTSMSLGTTR” replaced the mouse MLKL sequence “ELSKTQNSISRTAKS”. This “humanized” mouse MLKL is phosphorylated by mouse RIPK3 and is also recognized by human p-MLKL antibody ab187091 (Abcam).