Like Father Like Son?

With reference to Never mind the quality, feel the width,

I sent an e-mail to Dr. Gautam Sethi on 24.11.11:
“On the Abnormal Science Blog I have been criticizing the way, you presented some Western blot results using just one actin loading control for up to 19 distinct proteins. Unfortunately, the materials and methods section of the respective paper did not contain sufficient instruction to fully understand the underlying methodology. Since I plan another posting on this topic, the blog readers and the scientific public at large would very much appreciate if you could provide the underlying protocol for this kind of experiment, specifically these informations would be helpful:
how many gels were run, how many blots were performed, how many stripping steps were included.”

A reminder e-mail also failed to obtain an answer.

Dr. Sethi has 57 joint publications with Professor BB Aggerwal, Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX.

Of note, Dr. Sethi appears to be using the Western blot methodology he got to know as a postdoctoral fellow in Prof. Aggarwal’s lab.

Since Gautam Sethi refused to respond to my questions, I asked Prof. Aggarwal for methodological details in regard to one of his recent article:
Park B, Prasad S, Yadav V, Sung B, Aggarwal BB.
Boswellic Acid Suppresses Growth and Metastasis of Human Pancreatic Tumors in an Orthotopic Nude Mouse Model through Modulation of Multiple Targets.
PLoS One. 2011;6(10).

“Specifically, Figure 1 C displays Western blotting with 8 distinct proteins and 1 β-actin loading control. The Materials and Methods section does unfortunately not go into details how Western blotting was technically performed.
I would be most grateful if you could answer these questions:
How many blots from how many SDS-PAGE gels were performed. Were single blots stripped and reprobed.”

On 27.12.12, I received this response:

“Fig. 1C is a result of 3 distinct blots. beta actin from only one blot is shown, however.”

This answer was striking since 3 distinct blots should also contain three distinct  loading controls.
This point of view is shared in an editorial published in Journal of Clinical Investigation: “My understanding of a loading control is that it represents an analysis of an irrelevant protein from the exact same gel lane to assess how much sample was loaded in that particular lane. This seemed to be an elementary governing principle to me, which was why I was somewhat shocked when we had an article that, among other problems, ran the loading control on a separate gel at the same time. To me, running a parallel gel, even if the sample run on the gel was an aliquot from the same tube, does not demonstrate equal loading of sample in the experimental gel. How does one control for variations in pipetting such small volumes? …”

It appears to me that in the lab of Bharat B Aggarwal where Dr. Sethi had been trained as a postdoctoral fellow, no stringent rules for the use of loading controls in Western blotting are followed.

Sethi G, Ahn KS, Pandey MK, Aggarwal BB.
Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NF-kappaB-regulated gene products and TAK1-mediated NF-kappaB activation.
Blood. 2007 Apr 1;109(7):2727-35.
Cytokine Research Laboratory, Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

Fig. 2

Fig. 2A indicates that two separate blots with two distinct actin loading controls were performed to investigate six proteins under varying experimental conditions.

So far, so good, but fig. 2B illustrates four blots with identical actin loading controls under exactly the same experimental conditions (TNF versus Celastrol+TNF treatment of cells). If cells were treated differently, however, actin bands cannot be identical.
On the other hand, I wouln’t be surprised if fig. 2A had been assembled from more than one blot.
In these time course experiments, however, protein normalization is particularly relevant which makes correct loading controls indispensable (see also here).

In the Aggarwal lab the presumed irregular use of loading controls appears not to be the only issue at stake. Science integrity authorities should scrutinize publications from this lab (PubMed lists a total of 531) for other possible irregularities as well:

Bhardwaj A, Sethi G, Vadhan-Raj S, Bueso-Ramos C, Takada Y, Gaur U, Nair AS, Shishodia S, Aggarwal BB.
Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-kappaB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells.
Blood. 2007 Mar 15;109(6):2293-302.
Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.

Fig. 1

Fig. 7

This above example was obtained from the Japanese investigative blog http://blog.m3.com/Retraction. Significantly, this blog has been discontinued soon after it helped to uncover presumably massive data manipulations in articles from the lab of Prof. Matsubara (here, here, here).

I am confident that the Abnormal Science will manage to survive long enough to document more deviances from Prof. Aggarwal’s lab. Stay tuned.