I am using MEF (From BL/6 mice) as a model. While testing cell death in experiments, I am using MEF with TNF (20 ng/ml), CHX (10 micrograms/ml) as a positive control for apoptosis (TC) and those two along with Z-VAD-FMK (50 micromolar) as a positive control for necroptosis (TCZ). Now, I am using these treatments for 18-20 hrs, as my experimental conditions are for that long. While doing the WB, I do see the p-MLKL band at or just above 50 kd (predicted size is 54kd) in one of my experimental lanes which was expected but also on TNF+CHX treatment but not with TCZ treatment. On the other hand, when I blot for p-RIP, I clearly see a 78 kd band with TCZ treatment but not in TC treatment or my experimental condition. Interestingly, I do see the same band when I treat the cells of my experimental conditions along witH Z-VAD-FMK.
My question is what could be the reason for not seeing the p-MLKL band in TCZ treatment and while I see that in TC treatment and not seeing the p-RIP treatment while clearly seeing the band with TCZ treatment?
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