Question on western blot picture - (Sep/04/2018 )

Hello all,

I am reading a paper that can be found in the attachment or here: https://www.cell.com/cell-reports/fulltext/S2211-1247(18)30643-0?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2211124718306430%3Fshowall%3Dtrue#secsectitle0020

A simple question on this paper =>

Picture 3 C : does the picture seem ok to you? 

Am I missing something here or is the left part of the picture rather strange compared to the right part?

They show Co-IP, but in the input sample you do not see a band in the Pkc1 lane which is weird in my opinion.

If it is the same blot then why is there no band? My only idea is that it is because Pkc1 is only present in such low amounts that you need to do an IP first to concentrate it more to be shown on a blot later. However it seems weird this is not mentioned in the paper + in picture 3A you  can clearly see a band in the Pkc1 lane, which was a simple "input" sample so not sure why they have no band in picture 3C.

I find this type of pictures/blots rather fishy. 

Any insights?

Or am I missing something crucial here? I just can not seem to figure this one out.

It is possible that they have mis-labeled the image. You don't always see a band on an input, and tagged proteins can be easily concentrated by tandem affinity purification (TAP).

However, I wouldn't expect them to do a TAP procedure on the input, this should be lysate only, no TAP.

If you are concerned - there are a couple of things you can do - contact the authors and see if they can clarify. You can also contact the editors and see if they can clarify (I would only do this if there were significant concerns about several parts of the paper) and finally you can post on Pubpeer and see if anyone else picks it up.

bob1 on Wed Sep 5 14:07:56 2018 said:

It is possible that they have mis-labeled the image. You don't always see a band on an input, and tagged proteins can be easily concentrated by tandem affinity purification (TAP).

 

However, I wouldn't expect them to do a TAP procedure on the input, this should be lysate only, no TAP.

 

If you are concerned - there are a couple of things you can do - contact the authors and see if they can clarify. You can also contact the editors and see if they can clarify (I would only do this if there were significant concerns about several parts of the paper) and finally you can post on Pubpeer and see if anyone else picks it up.

I understand you do not always see a band on an input, but the thing is: you do see a band in the image 3A , so I do find it a bit strange.

Ok , I admit, it seems they did use the whole protein for 3A which is more than the "input" for 3C if they used the same "input"as for the Co-IP. But still, I find it rather odd.

And mis labeled the image? if this is the case, then something fishy is going on since then you have a problem explaining the CO-Ip result. 

Oh I tried to contact the authors, 3 of them actually, but none of them have replied....

I have no real problems with the rest of the paper to be honest, I am strictly interested in this part of the experiments.