im studying the signaling response as well as gene expression response upon applying mechanical stress on cancer cells.
Generally, signaling response do not take long to be activated/deactivated as signaling pathways can have activity altered by phosphorylation (phosphorylation cascade - in which one signaling protein phosphorylate the other setting off chain reaction). I have been looking at western blot analyses and identified some MAPK pathways as being implicated - again i applied stress at various short time durations below one hour (5 mins, 15 mins etc). It is safe to say that my conclusion is such that mutiple MAPK pathways such as P38 MAPK is activated.
I then proceed to do RNA-seq which my main aim is to look at global gene expression changes - and since most gene expression studies involve putting in stimuli for longer periods of time then those used in studying signaling pathway activation - i applied the mechanical stress for 6 hours. All seems fine until i did the pathway enrichment step in which i discovered none of the MAPK signaling pathways(ERK, p38 etc) are enriched. I am aware that these pathway enrichments rely on the fact that the protein is overly expressed but that does not mean that their activity is not altered. How do people interpret signaling pathway analyses from gene expression studies? Can they say that ''this pathway is activated, since it is enriched for genes that were upregulated'' . what about those pathways that were not enriched - are these genes ''not activated'?