I would like to test if 1 of intracellular bacterial effectors dephosphorylate p44/42. I read a lot of articles and confused. Do I have to starve cells for serum? Do I have to do infection experiment without serum? I also found that peoples used PMA to activate phosphorylation and in mutant and wt (salmonella). But they did not mention about serum. As well I am having hard time to detect p44/42 as well. Can you help me with this?
Well...I do not have expertise in bacteria but in mammal cells its easy to activate and deactivate ERK1/2. If cells are cultured in serum suplemmented medium you might find elevated levels of phosphorylated ERK1/2. So I always serum starved cells for an o.n. before stimuli. For ERK1/2 stimulation you can use PMA or simply 10% of serum. Usually this kind of stimuli leads to an ERK1/2 phosphorylation rapidly (within 1-2 min) that descreased to basal levels in 15-30 min. I think you should get a time-course for ERK1/2 activation in your cell system using PMA or serum to find the better time to cell lysis. I feel that addiction of the intracellular bacterial effectors that you want to test should be added 30min before stimuli as we do when using ERK1/2 inhibitors but you need to test this times.
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