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Inhibition of a protein upstream in signaling cascade ?


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#1 Tabaluga

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Posted 17 March 2013 - 04:25 PM

Hi people,

let's say I have a protein Y to which there is no known inhibitor, and I still want to study the effect of inhibiting it on a certain cellular process (not considering the possibility of doing a knockdown). There is however an inhibitor available of another protein X upstream in the signaling cascade, which regulates my protein Y. So if I were to inhibit the upstream protein X and could show at the same time that levels of my protein Y go down and the expected cellular process stops, would you regard this as at least a strong hint (doesn't prove anything obviously) that my protein Y is associated with this process ? Or would you say this experiment is a waste of time since the upstream protein X obviously regulates many other proteins too and thus it is "guilt by association" only ?

I'd very much appreciate your comments on whether this approach makes any sense (personally, I must say I'm not convinced), or if you have any other ideas.
Thanks!

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#2 jerryshelly1

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Posted 17 March 2013 - 08:02 PM

You can. Whenever you inhibit a protein you should definitely consider all of the indirect effects it is having on your cell system. It wouldn't hurt to try if it is within reason of your PI's budget.

What is your reasoning for not wanting to knock your protein out? My favorite approach is when a paper knocks out a gene and then transfects an inducible promoter in a cell line. By adding your inducer, you can control your protein expression and look at the direct and indirect effects of its expression. Just something to consider. I do not see any harm in your method, unless it would kill the cell... Does any previous literature explain inhibition of that particular protein in your current cell line?

#3 Tabaluga

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Posted 18 March 2013 - 05:21 AM

You can. Whenever you inhibit a protein you should definitely consider all of the indirect effects it is having on your cell system. It wouldn't hurt to try if it is within reason of your PI's budget.

What is your reasoning for not wanting to knock your protein out? My favorite approach is when a paper knocks out a gene and then transfects an inducible promoter in a cell line. By adding your inducer, you can control your protein expression and look at the direct and indirect effects of its expression. Just something to consider. I do not see any harm in your method, unless it would kill the cell... Does any previous literature explain inhibition of that particular protein in your current cell line?


Thanks for your comment. I agree knockdown is the best way, but is currently not an option for us for time and project management reasons. The effects of inhibition of my protein of interest are not known in literature, while inhibition of the upstream protein (PI3K, actually) is described.

Any further opinions ? Or problems someone sees with the approach ? Because the last thing I want to do is evoke the impression that I am suggesting an unreasonable and not thought-over experiment ... Posted Image

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#4 ravibiosciences

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Posted 24 March 2013 - 04:12 AM

X
|
|
|
Y

To know the best possible signalling mech in any pathway is to use a) Pharmacological inhibition or B). RNA interference. You have no drug which inhibits your X and you don't want to work with siRNA exp's either.
Two questions i would expect and look for a convincing answer.
1. I guess you might have already considered over expression of your X to find out or rule out the functional effects leading to your hypothesis via "Y"? (of-course, with proper +/- controls)
2. Is there any stimulant that you can show direct regulation of your ''Y'' irrespective of your upstream targets?

If you want to play with upstream targets, I am afraid to get increased impact of your paper in a very short time Posted Image .

Good luck and enjoy doing science.

#5 Tabaluga

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Posted 24 March 2013 - 04:38 AM

Hi,
thanks for your input ! I don't have a stimulant to Y either. I don't quite understand what overexpression of X could give me - we already know it is definitely a regulator of Y.
Also, what do you mean by this sentence ? I'am afraid I didn't get it. Posted Image

If you want to play with upstream targets, I am afraid to get increased impact of your paper in a very short time Posted Image .


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#6 ravibiosciences

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Posted 25 March 2013 - 01:12 AM

As you said you have very short time. I mean to say, it might take long time than you expected to get it published Posted Image




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