Anti-MLKL Antibody

To test the apoptotic, necroptotic, and authopagic protein targets implicated in human normal fibroblasts treated with 3-MA, Western blotting was performed. Briefly, cells were seeded in 2% FBS medium overnight. The cells were treated with 1 mM 3-MA for 4 hours, and the cells were collected and lysed with the buffer [1% SDS, 10 mmol/L Tris-Cl (pH 7.6), 20 μg/μL aprotinin, 20 μg/μL leupeptin, and 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl fluoride]. The protein concentrations were determined using the Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, IL). Fiften micrograms of protein were separated on SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with the appropriately diluted primary antibody at 4°C overnight. After washing with TBST, the membranes were incubated with appropriately diluted HRP-conjugated secondary antibodies at room temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotechnology, Inc., Piscataway, NJ).