Hi. With phosphorylated proteins, you might consider using phosphatase inhibitor - we added calyculin A (final total concentration of 100 nM in cell medium) onto cells 30-60 min before lysing cells. It did not seem to affect cell viability.

It could be a calpain/cathepsin/granzyme target or loaded in exosomes to sent out. Think of secondary necrosis/necrosis if cell death is not prevented.

 My deep thanks Dr.Lavogina and Dr.Jinesh.

ASKM,

First, you must consider that the caspase inhibitors actually inhibits the apoptosis induction and not all the processes upstream of the caspase activation. For example, We used to get cytochrome c release even in the presence of Z-VAD-fmk, but there was no activation of caspases and also the cell death was inhibited. We finally realized (and proved) that cyt-c release is an upstream event to caspase activation and ROS can even directly release the Cyt-c from mitochondria by altering the oxidative status of Cardiolipin. So you may be actually getting some good results. Instead of trying to prove the other things around, just follow the results you are getting, and look for the mechanism involved. You have already repeated the experiment 4 times, so definitely there is nothing wrong with your experimental setup. Go ahead and explore the other possibilities, Good Luck

My deep thanks Dr. Ishaq. By the way our drug also is supposed to activate ROS production but we did not check that yet in our model.