Research integrity

On Western blot loading controls: lessons from Richard Moriggl lab

Western blot, a method to separate proteins by size and analyse their relative expression levels, is a much maligned technique of molecular cell biology. The website PubPeer is flooded with evidence of manipulated Western blots, where gel lanes were inappropriately spliced, or where bands digitally duplicated or erased. Some even question the technology as such, since it is indeed mostly Western Blots (and other gels, like Northern Blots for RNA or Southern Blots for DNA or RNA PCR-amplification gels) which are flagged for image manipulation.

It is however not the technology to blame for all the rigging done with it, but the simple fact that it is image based. Anyone with a minimum of image analysis skills or a good eye for duplications can spot Western Blot manipulations. You do not need to be an expert in the technology or even a biologist, to find data rigging. This is exactly why a certain aberration in Western Blot integrity is often either overlooked or dismissed as incompetent nitpicking: the absence of proper loading controls. Just like it seems to be occasionally the case in the publications from the lab of Richard Moriggl, director of the Ludwig Boltzmann Institute Cancer Research and professor at the Medical University of Vienna, Austria. The Moriggl lab studies molecular signalling in cancer cells and tissues, hence its focus on the analysis of regulatory protein phosphorylation and how it changes under various conditions. Such analysis must be always supported by proper gel loading controls, which seems not always be the case here. A reader of my site contacted me with some examples of such inappropriate gel presentation in Moriggl papers, some of which he already posted on PubPeer.

Do the bands of total protein AKT match those of phosphorylated (pAKT) in this Moriggl paper Mueller et al, Diabetes, 2017? It seems they do not, which indicates the two separate gels are presented as one.  The paper is so fresh, original data must exist to answer the questions. Source: PubPeer

There, a number of Western Blots for a phosphorylated protein seem not to fit the corresponding loading controls provided in the figure. It is not a small issue, because the purpose of such gel analysis is to compare the levels of a certain phosphorylated protein among different samples, say control versus various treatment or a the effect of a treatment at different time points. This is why it is important to ascertain that exactly same amounts of protein-containing cell lysates are present in each gel lane. Here, one does not rely solely on prior protein concentration measurements, but also performs loading controls of the same gel, for a protein which levels are not expected to change in the course of treatments. In the case of phosphorylation analysis, one usually probes with specific antibodies for the same total, non-phosphorylated protein. Only when the equal loading is ascertained can one compare the changing levels of the phosporylation of your protein of interest, or in fact of the overall expression of any other protein you wish to analyse, using corresponding antibodies.

Key point here: it must always be the same gel. It is of no use to detect amazing differences in a protein expression or phosphorylation, while not having a proof that the gel was indeed equally loaded. To probe a different gel where same samples were re-run for an allegedly corresponding loading control does not really make much sense: no two gels are ever exactly same, and a lot of incidental and not so incidental mistakes can happen during the sample loading or the gel run or transfer. This is also why one can spot if the two images really belong to the same physical gel: the lane widths and band sizes and shapes are a give-away if they do not match. Indeed, the sacked cheater Irina Stancheva was caught not only on gel band duplications, but also on using inappropriate loading controls.

FIG B
In Kaltenecker et al Diabetologia, 2017  , blots for p-AKT and Akt do not seem to match, does the panel HSC70 belong to any of these 2 gels, or is it a 3rd one? the question is raised for both panels, “Liver” and “EWAT”. Comments here and in other figures are from the reader, who provided me with those.

Yet the loading controls are often seen as irrelevant. The fallen star of plant sciences Olivier Voinnet built his misconduct defence on his having used a loading control library, where he pulled out a gel image to fit the analysis he aimed to publish. A loading control duplication in a paper from Ronen Alon’s lab at Weizmann Institute was also explained with the use of such libraries (see here).  In any case, even if the loading control panel is found to be photoshopped or duplicated, even some established scientists will object: it is just a loading control! The main analytic Western Blot panels are perfectly fine! This is however absolute nonsense, from the scientific point of view.  Every Western Blot analysis needs a loading control, done for exactly same gel. Otherwise the entire protein expression analysis becomes utterly useless and one can skip using figures in scientific publications altogether.

fig-c.jpg
Also in Themanns et al Sci Reports, 2016, HSC70 served as loading control. But the bands appear too different for it to be the same gel.

Moriggl never replied to my emails. Hence I contacted the Medical University of Vienna with the evidence in his papers, in August 2017 (incidentally, one member of their Scientific Advisory Board is the radiologist and self-plagiarist Hedvig Hricak, from Memorial Sloan Kettering Cancer Center (MSKCC) in New York, read here). The Vice-Rector for Research, Michaela Fritz  replied to me the next day:

“Thank you for the information. After consultation with the University of Veterinary Medicine Vienna, the case will be taken up by the Ombudsman of the Vetmeduni, which has already contacted you”.

Indeed, the Ombudsperson of the Vetmeduni Vienna, Mathias Müller wrote to me in parallel:

“Your mail from 23 August 2017 to the management of the Medical University of Vienna was forwarded to the Ombudsoffice of the Vetmeduni. The issues are currently being discussed with the authors. The PubPeer presentations will be addressed with the publishers by submitting point-to-point comments”.

FIG D

In Javeheri et al Cell Death & Disease 2016, blots for signal HA-EWS/FLI1 and HSC-70 (upper figure) as well as for phosphorylated protein RB (pS780-RB) and total protein RB (lower figure) seem not to match. These gels seem to lack their correct loading controls.
The mismatched phospho- and total protein Western blots are not the only problem with this Javeheri et al 2016 paper from the Moriggl lab. Another loading control blot seems to be duplicated (Source: PubPeer), which should be easily checked given the recent publication of this paper and certain availability of original data.

Prior to writing this article, I asked the Ombudsman Müller for an update, but received no reply.  There are more inconsistencies in Moriggl papers, like this collaborative paper Gotthardt et al Cancer Discovery 2016 from the neighbouring Vienna lab of Veronica Sexl at the same Medical University Vienna. Interestingly, Sexl has other papers flagged on PubPeer and had already to correct one, namely Kollmann et al, Cancer Cell 2013.

FIG G
The loading control for total protein STAT5A/B does not seem to match its phosphorylated blot in   Gotthardt et al Cancer Discovery 2016

It is of course very likely that correct loading controls will be found and Moriggl will be able to correct his publications to show the matching pairs of gels. Those papers are very fresh after all, the data is certainly readily available. Otherwise readers would be forced to be sceptical about the scientific validity of these Western blot analyses. Without the correct loading control for the exactly same gel, to show the protein loading ratios, a celebrated difference in expression or phosphorylation of a regulatory protein might be nothing but an artefact.


Update 2.11.2017. Matthias Müller, ombudsman of the Vetmeduni Vienna, wrote me this email today:

“Dear Dr Schneider!
With regard to your e-mail dated 23.Aug.2017 to the Rectorate of the Medical University of Vienna, which has been forwarded to the Ombudsman of the Vetmeduni, I can inform you that the issues were clarified with the authors of the publications in Cell Death & Disease and Diabetes.
Replies were made to the PubPeer posts by means of ‘point-to-point’ statements and also sent to the editorial boards of Cell Death & Disease and Diabetes with a request to consider a publication of a corrigendum.
Both Editorial Offices have now approved the Erratum requests and will publish the Corrigenda in the earliest available journal issue”.

Update 13.11.2007. Is the Moriggl lab under investigation by Austrian Science Fund FWF?


 

 

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57 comments on “On Western blot loading controls: lessons from Richard Moriggl lab

  1. Rex Rictor

    Antibodies are a disaster, immunoblotting as well. In particular for phosphorylation. Mass-spec is the way forward, its not trivial but Ab’s simply suck for detection/measurement purposes.

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    • Adrian, set up you own blog and rant there, as I did when Ivan Oransky of Retraction Watch started to teach me how Western Blots work.
      And no, you cannot copy-paste your lectures, pardon comments on every article on my site which mentions western blots.
      Finally, flattered as I am about your assumption that I am the dark eminence of all science, pulling strings and ordering about universities and publishers, that is actually not true. As I suggested before, discuss it for example with Nature, they post such blot guidelines on their Instructions for Authors page.

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    • You have written several blog posts on this topic, that to your credit have been widely picked up. Like it or not now, you have taken a lead on this, made accusations, and promoted a non-evidence based doctrine for how western blotting should be performed. That is the position you have got yourself in to, that is not my fault, we are allowed to challenge you.

      And isn’t it ironic that when someone tries to hold you accountable you deflect, dismiss and ignore. You should be held accountable, just as you so love to do to everyone else.

      Science is about challenging evidence and opinions. Isn’t that your supposed occupation? Why can’t you discuss any of the science and evidence, and why do you want to ignore my evidence-based opinion on this website?

      Is it because I’m making sense and you are too proud to climb down from your position?

      Or do you really think all of these papers (PMIDs: 25540176; 24738055; 25852189; 30800670; 25059473; 23709336; 24023619; 18571732; 21186791; 23454168; 24561642) showing that loading controls cannot typically be reliably performed on the same membrane as lower expressed proteins of interest, because their linear ranges are different, are wrong?

      Are you calling for the correction of retraction of thousands of life sciences papers?

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  2. To APR (also as Coffea Betamponensis from PubPeer): The original paper cited a reference (Number 21, Direct glucocorticoid receptor–Stat5 interaction in hepatocytes controls body size and maturation-related gene expression) about Western blot technique. That paper states that “blots were stripped and reprobed”. The correction admits that the blots were run in parallel, and explicitly states that the new blots were probed on the same membrane. However, in the corrected figures, pAKT-AKT-HSC70 bands still lack similarities in shape and running direction, just as in the original paper. If it is so accepted to run parallel blots, why the Authors did correct their paper at all?

    Like

    • APR replied to you with a simple copy-paste of this lecture, again:
      https://forbetterscience.com/2017/10/18/on-western-blot-loading-controls-lessons-from-richard-moriggl-lab/#comment-55122
      This is too silly. I am deleting redundant copy-paste comments.

      Like

      • Mark K. Trent

        This reasoning is the classical bull++++++ing: let´s talk about something else and never answering the questions.
        Ho-Ye: the paper you have mentioned, contains blots with possible non-indicated splicing.

        Like

    • If you wish to analyse many proteins, you must indeed run several parallel gels. Each gets proved for equal loading, either Ponseau S or actin, tubulin, etc.
      See how simple it is? You lengthy straw man arguments are getting boring.

      Like

    • Again, you either haven’t read, or understood my post, or are deliberately trying to protect your position.

      How do you propose doing an actin or tubulin blot on every membrane? I have repeatedly explained why this is flawed and in most contexts meaningless. This is probably the key take home message!! It clearly isn’t so simple for you otherwise you would have got it by now.

      Before you write blog posts about loading controls again, really think through what you are writing.

      Do you really think all of these papers (PMIDs: 25540176; 24738055; 25852189; 30800670; 25059473; 23709336; 24023619; 18571732; 21186791; 23454168; 24561642) showing that loading controls cannot typically be reliably performed on the same membrane as proteins of interest (e.g. AKT), because their linear ranges are different, are wrong?

      But total protein stain, yes! We are making some progress. That is the future, no more meaningless single loading controls on every blot that you are still (!) promoting.

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      • You don’t even understand why Ponseau S is more superior. It’s because it shows full gel. The red bands you see on Ponseau S are those of highly expressed proteins, like those used for loading control. The rest is just a pink blur. Basically Adrian, you have no clue what you are talking about. But you do love hear yourself talk (metaphorically here), yet please stop right now. This comment section is not your personal lecture theatre. Thanks and goodbye. You know how I mean it.

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    • Your complaints about Figure 6E in https://forbetterscience.com/2018/12/06/western-blot-loading-control-libraries-and-beyond/ perfectly illustrate your confusion.

      “New gels in Figure 6E are hopelessly overexposed [GAPDH], neither blot background nor any baseline trace signal of phosphorylated AKT is visible (AKT-expressing samples without obvious phospho-AKT signal should at least show miniscule trace bands when residual signal is digitally boosted). This blot overexposure practice is not acceptable in 2018 anymore”.

      You are expressing annoyance at the very same problem you want to export to all scientists – more often than not you cannot expect GAPDH and p-AKT to be in their linear signal ranges on the same blot (PMIDs: 25540176; 24738055; 25852189; 30800670; 25059473; 23709336; 24023619; 18571732; 21186791; 23454168; 24561642).

      GAPDH has a 500-2000-fold the copy number of AKT, and much much higher for p-AKT (At “baseline”, 100,000-fold? 1,000,000-fold?).

      You cannot perform a GAPDH blot on the same membranes as you demand and expect it to be meaningful.

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  3. Here is the PubPeer link to the paper which describes how the AKT-pAKT WBs have been performed in the Diabetes (2017) article discussed above: https://pubpeer.com/publications/7438809278C6E0BAE776D00CDF388D#1

    (Yes, you see it well, the blots do not show pAKT or AKT.)

    Like

  4. I can see what you are saying APR. I personally have always been suspicious of the usual loading controls, the signals are more often than not far too strong and saturated to be good controls. Obviously then doing that type of control on every blot with the same protein ug only just multiples the artefacts you get…

    Perhaps Leonid you could edit your articles to focus solely on clearly unacceptable practices and leave out the “loading control libraries” stuff as it only reinforces this problem?

    Like

    • There is Retraction Watch, where nobody will ever discuss a loading control or point fingers at people one must never point fingers at. They also know how to science properly, those Watchdogs.

      Like

  5. Jablonka

    A new comment (comment 27) in this thread shows that loading control is misleading. And stripping the membrane too. https://pubpeer.com/publications/2D084E91EDC85D6C23BC63ACF5E6A8#27
    Any opinion?

    Like

  6. Loading control is just a “mantra” and an “inferior technique” according to this thread of discussion: https://pubpeer.com/publications/2D084E91EDC85D6C23BC63ACF5E6A8

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  8. The problematic Western blots have earned a retraction at Cell Death and Disease. https://doi.org/10.1038/cddis.2016.268 “The authors have retracted this article because in Supplementary Figure 8b (1) the left-hand and right-hand MCL1 blots are the same, and (2) the cleaved caspase-3 blot lane 5 in the left panel is a duplicate of the first lane of the cleaved caspase-3 blot in the middle panel. Authors M. Hofbauer, M. Wiedner, C. Hartman, J. Tuckerman, J. Pencik, E. Tomazou, M. Schlederer, T. Javaheri, M. Mikula, H. Nivarthi, L. Kenner, D. Aryee, B. Sax, F. Grebien, H. Dolznig, G. Richter, M. Logan, B. Maurer, A. Üren, H. Kovar, R. Morrigl, and H. Pham agree with the retraction. Authors Z. Kazemi, M. Kauer, and R. Noorizadeth have not responded to correspondence about this retraction. The authors are repeating their experiments and a new manuscript will be submitted for peer review.”
    It was gentle from the editors to allow an author retraction (instead of an editorial retraction). Looking forward the new manuscript. I wonder, whether the host institutions have been alerted and they intend to inspect the work of these labs. Also, do the funding agencies know about the case?

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  14. The issues with one the here discussed articles have been investigated by its funding agency (German Research Foundation). The conclusion is that all was fine with the Western blots. Das Erratum zu dem Diabetes Article (Kristina M. Mueller, Kerstin Hartmann, Doris Kaltenecker, Sabine Vettorazzi, Mandy Bauer, Lea Mauser, Sabine Amann, Sigrid Jall, Katrin Fischer, Harald Esterbauer, Timo D. Müller, Matthias H. Tschöp, Christoph Magnes, Johannes Haybaeck, Thomas Scherer, Natalie Bordag, Jan P Tuckermann and Richard Moriggl, Adipocyte Glucocorticoid Receptor Defi-ciency Attenuates Aging- and HFD-Induced Obesity and Impairs the Feeding-Fasting Transi-tion, Diabetes 2017) enthielt keinen Hinweis auf eine Duplikation.
    Das Verfahren ist damit abgeschlossen.

    Mit freundlichen Grüßen

    Kirsten Hüttemann
    DFG

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  15. Das Erratum zu dem Diabetes Article (Kristina M. Mueller, Kerstin Hartmann, Doris Kaltenecker, Sabine Vettorazzi, Mandy Bauer, Lea Mauser, Sabine Amann, Sigrid Jall, Katrin Fischer, Harald Esterbauer, Timo D. Müller, Matthias H. Tschöp, Christoph Magnes, Johannes Haybaeck, Thomas Scherer, Natalie Bordag, Jan P Tuckermann and Richard Moriggl, Adipocyte Glucocorticoid Receptor Defi-ciency Attenuates Aging- and HFD-Induced Obesity and Impairs the Feeding-Fasting Transi-tion, Diabetes 2017) enthielt keinen Hinweis auf eine Duplikation.

    Das Verfahren ist damit abgeschlossen.

    Mit freundlichen Grüßen

    Kirsten Hüttemann

    Like

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