I am growing THP-1 cells. After 48 hours of PMA exposure (50ug/ml) and two days of rest with RPMI 1640 medium with 10% FBS I see phosphorylation of the P65 subunit of NFkB (without any stimuli such as LPS added). If I add LPS I see even more phos of P65 (expected). Why am I seeing NFkB activation in these cells in baseline condition without LPS?
THP-1 cells are monocyte in nature which differentiates into macrophages by PMA stimulation. PMA affects the activation of protein kinase C and first stops the cell proliferation and then starts cell differentiation process. NFkB is one of the transcription factor involved in the differentiation event. Probably because of this you are getting the NFkB expression at basel level.
You may not have a long enough rest time, and the macrophage population may have some transient expression in a sub-population that you cannot discriminate by WB. Have you tried to detect p65 translocation by immunofluorescence to see if all of the cells are activated, or just some? You could try to bring the serum down to 0-0.5% as well, as serum components can transiently activate NFkB under certain conditions.
As Jordan mentioned above, serum can have effects on most of the signalling pathways and you may have to serum starve (reduce serum concn) the cells to reduce basal phosphorylation events. Even a fresh medium change (with serum at the same concentration as before) can induce NFkB signaling. If you are specifically interested in LPS-signaling and if LPS is stimulating the cells significantly, it is better not to change anything.
If you change the medium before treatment you should wait at least 24 h before LPS treatment to avoid medium/serum effects.
Thanks Jordan R Yaron....
I tottaly agree with you...
Serum components does have eefect on intracellular signalling and phosphorylation events ...
which could be one of the reason for getting such NFkB expression in macrophages....
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