I work with ER stress in primary cultures of cardiomyocytes. I have checked induction of GRP78 and other ER stress markers by both realtime PCR and western blotting. In spite of induction of gadd153 and XBP1 splicing, I do not see any induction of GRP78 in the western blot, although I see an upregulation with thapsigargin. Could anyone explain why this is happening? I have tried antibodies from different companies.
In addition to other suggestions already made, you should be aware that differences in protein levels (induced or steady-state) are rarely as high as differences in corresponding mRNA levels so don't be alarmed if you find BiPmRNA is elevated in but BiP protein is not. I'm unaware that this generality has ever been documented; this is based on 25 years experience evaluating mRNA and protein expression of a large number of different types of genes/proteins including BiP. Do take note of Jay Brewster's comment about negative feedback control of BiP translation, although I think that induction of ER stress was shown to override this feedback control.
I think you should make sure that the mRNA level of GRP78 gene upregulated is real, and if this is true, is your antibody is really work, if all these are no problem, in some condition some gene really could be detected the changes in mRNA level, but could not be detected any change in protein level. As I knew, in mouse neutrophils, CD115 gene could be detected at mRNA levelbut could not be detected at protein level in neutrophils,
GRP78 protein levels have been noted as less dynamic than other markers for ER stress. Some cells seem to show good upregulation along with other ER stress markers, while others show only marginal changes in total protein level. I would believe your results if you have good antibody and a clear band on your immunoblot. We've seen good mRNA induction (~5 fold) with cells only showing perhaps a 2-fold increase in total GRP78 protein. This may be due to the instability of some ER localized proteins during ER stress (increased ERAD). If you are simply assessing indicators of ER stress, you might look at GRP94, which often is more dynamic. Perhaps the most sensitive marker for ER stress is the PCR based XBP1 splicing assay. That assay works quite well and is very sensitive.
Could it simply be part of a stress response where some protein functionality is attenuated under stressful conditions? If so, perhaps if you vary the stress levels (like titration) and see if the protein function is restored at lower stress levels, you may have something important here...a modulation of function and a marker for levels of cardiomyocyte stress.
I believe your results.
I have a few suggestions,
Might be the protein is unstable, so use TCA precipitation.
It might be possible that the expression is regulated at the level of translation, so you need to check if there is a possibility for the formation of hair-pi loops upstream to ORF.
ANother possibility is the presence of short uORF that regulate the expression.
Another possibility, after checking any technical issue, of course, is that the mRNA levels are upregulated in roder to maintain the same protein expression. This would happen if GRP78 degradative pathway is enhanced.
Thanks a lot for all your replies.
Prof. Brewster, I will be grateful if you could please suggest me some articles that explain this about GRP78.
This article might shed some light on the issue. Others have mentioned this in passing (once mentioned in the comments to me from review of a grant application)
BiP is feed-back regulated by control of protein translation efficiency
June 1, 2002 J Cell Sci 115, 2443-2452.
I have been working with BiP since three years and I want to share my experience: BiP as well as GRP94 and other chaperones are very long-life proteins, so it means that it may be hard to find more than 2-3 fold induction at protein levels. So I don't think you did something wrong in the preparation or analysis of your samples. And also it depends a lot on the duration of the treatment. We published recently a paper about that, take a look to our western blots and RT-q-PCR, where we used different treatments and different time points.
In addition to other suggestions already made, you should be aware that differences in protein levels (induced or steady-state) are rarely as high as differences in corresponding mRNA levels so don't be alarmed if you find BiPmRNA is elevated in but BiP protein is not. I'm unaware that this generality has ever been documented; this is based on 25 years experience evaluating mRNA and protein expression of a large number of different types of genes/proteins including BiP. Do take note of Jay Brewster's comment about negative feedback control of BiP translation, although I think that induction of ER stress was shown to override this feedback control.
Thank you for your suggestions and comments Ms Eletto and Prof. Cavener
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