The bands of WB suggesting the natural compound inhibited the phosphorylation of both Stat5 and Stat3. The extent of reduction of p-Stat3 and p-Stat5 is different, the level of p-Stat5 reduced more remarkable.
So, the questions is, can we just assume that the target of this compound is more likely to be Stat5 or the upstream of Stat5. Or in other words, this compound is more easily combine with Stat5 or the upstream protein of Stat5? If so, I want to know what methods we can use to further prove my assumption.
When looking at STATs phosphorylation status is no longer a suffice readout for activity. The best way to prove an effect is my doing qPCR for target genes activated by stat5 and 3 , to show specificity towards STAT5. For upstream you can try probing for phospho and total levels of the IL2 receptor /Jak pathway.
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