promoter regulation but not protein regulation - (Aug/29/2007 )

Hi!

I'm studying the role of MEK/ERK pathway on p21 expression, using a p21-luc construct. I've observed that, when I transfect my cells with a MEK constitutively active, p21 promoter activity decreases. However my surprise was that when I analize p21 protein levels by western blot I do not observe any diference between control and cell with MEK c.a. So, is it possible that MEK/ERK is downregulating p21 promoter but this process hasn't got effect on protein levels?

Thank you very much for your help!!

Have you taken a possible time shift between promoter acitvation and protein expression level change in account? perhaps you should add some hours to the time when you look at your promoter, so like 12h after and then compare your p21 expression of constitutive active cells against normal cells.

it's also possible that your p21-luc construct does not contain all the regulatory elements, maybe you should try to confirm this by qPCR on endogenous p21 mRNA?

Hi,

I posted this previously but it got deleted for some reason (why?).

First check to see if the mRNA level is changed by the MEK hyperactivity.
Regulation of luciferase is not necessarily the same as a gene in the context of chromatin.
USe the luciferase as a follow up to define the sequence that may be affected by the MEK.

It is possible that p21 is highly regulated at the level of translation so that the mRNA goes down but the protein will not.
(This is often true for p53).
Also, use MEK inhibitors to see if p21 mRNA is affected.

Hi!

Thank you for your help and your advices!

I'm trying to analize p21 levels 12 h after luciferase assay. I hope it's enough time for translation of new mRNA.

I'm also quantifying p21 mRNA to make sure that the decrease of luciferase activity can be extrapolated to p21 gen in chromosome.