hi everyone, I am working on the interation between a viral protein A and host protein B. I found that this viral A protein inhibits mRNA expression of host B protein by real time PCR. As always, I cloned the promoter of B (1.5kb upstream of TSS) to a Luc reporter and transfected it to a B-high-expressing cell line. however, I did not see any inhibition. Although it looks like this reporter might be OK because I saw a 10-fold increase compared to the Luc reporter without insert. I dont have a reliable positive control but I used chemical treatment which I have confirmed to downregulate B mRNA expression. This chemical also did not inhibit the promoter activity. Instead, both induced a bit increase.
I know it is still likely that my promoter reporter was not good. But what if it was, then what exactly downregulated the mRNA of B. Do you have any idea regarding the regulation of mRNA? Any comment would be appreciated. Thanks.