Reactivity of vertebrate-directed phospho-eEF2 antibody against the Caenorhabditis elegans orthologue phospho-EEF-2 [version 1; peer review: 2 approved]

Eukaryotic protein translation is divided into three mains stages: initiation, elongation and termination. Regulation of this process occurs at the initiation and elongation step. eEF2 kinase phosphorylates eEF2 factor, blocking its ribosome interaction and thus translation elongation. This kinase activity can be detected by measuring eEF2 phosphorylation status. Here I show that vertebrate-specific antibody against phospho-eEF2 has excellent reactivity against C. elegans orthologue protein phospho-EEF-2.


Introduction
Protein synthesis determines the cellular proteome and its regulation is pivotal to maintaining cellular homeostasis.This process is divided into three mains stages: initiation, elongation and termination 1 .The most studied step in eukaryotes is translation initiation and its regulation is critical to cell survival under stress conditions.Elongation regulation is also important in modulating translation.During this process, eukaryotic elongation factor 2 (eEF2) catalyzes the translocation of peptidyl-tRNA from the A site to the P site on the ribosome.The phosphorylation of eEF2 at threonine 56 by eEF2 kinase (eEF2K) inhibits its binding to ribosome and thus its activity [2][3][4][5] in stress-or starvation-related conditions [6][7][8] .eEF2 kinase is an atypical α-kinase, normally dependent on Ca 2+ ions and calmodulin, and apparently has only one substrate, the elongation factor eEF2 9 .eEF2 phosphorylation by eEF2K results in a reduction in translation rate.C. elegans processes eEF2 and eEF2K orthologues, named as EEF-2 (94.8 kDa) and EFK-1 (87.8 kDa), respectively.As in other eukaryotes, the Thr56 residue and adjacent sequences in EEF-2 are conserved.The only described data about efk-1 shows that it is important to nutrient deprivation resistance 10 .My lab studies the role of efk-1 in various aspects of C. elegans survival and the simplest way to measure its activity is to determine the EEF-2 phosphorylation status.Here we show that phospho-eEF2 antibody from Cell Signaling Technology Inc., specific to vertebrates, has excellent reactivity against C. elegans phospho-EEF-2 and this property is preserved after freeze/thaw cycles.

Results
Using the described protocol, I could specifically detect the phosphorylated form of EEF-2, eEF-2 orthologue in C elegans since in efk-1 knockout worms, used as a negative control of efk-1 activity, there is no equivalent signal in western blots (Figure 2 and Figure 3).Detection of α-tubulin was used as a western blot load control.Some differences can be observed in α-tubulin detection when comparing lanes (Figure 2 and Figure 3), despite the use of approximately the same number of nematodes.This can be explained by the fact that some worms remain attached to the pipette tip after washes, and this effect can be circumvented adding 0.01% Triton X-100    (Sigma Aldrich) to C. elegans wash buffer (M9 buffer).In addition, both antibodies (directed to target and load control) can be used and detected simultaneously, reducing analysis time (Figure 3).
EEF-2 phosphorylation is not observed in efk-1 knockout C. elegans, indicating that EFK-1 is the sole EEF-2 kinase in my tested conditions.Surprisingly, primary antibodies diluted in TBS-T-BSA (either phospho-eEF2 and α-tubulin, together or individual dilutions), stored at -20°C, can be reutilized at least five times (by thawing at room temperature) without losing specificity/reactivity (Figure 2B).

Conclusion
In this work, I show that vertebrate-directed phospho-eEF2 antibody specifically recognizes C. elegans orthologue phospho-EEF-2.This finding is very useful to those who work with translation elongation regulation using C. elegans as a model and I recommend this antibody to detect EFK-1 activity in C. elegans.

Competing interests
The author declares no competing interests.

Grant information
This work was supported by CNPq -Centro Nacional de Desenvolvimento Cientifco e Tecnologico, Capes -Coordenaçao de Aperfeiçoamento de Pessoal de nível Superior, and PRPq-UFMG -Pro-reitoria de Pesquisa da Universidade Federal de Minas Gerais.
I confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Viviane Alves shows that a phospho antibody raised against mammalian eEF2 can specifically detect the phosphorylation status of its counterpart in C. elegans, EEF-2.This is conceptually a very important validation in the field, especially because antibodies produced to detect specific phosphorylation sites in mammalian cells very often do not cross-react with non-mammalian organisms.The challenge in making use of these antibodies is that successful detection may depend on the specificity and affinity of the antibody for the phospho-protein of interest and on the amino acids sequences surrounding the specific phospho site.To make matters worse, the sequence and size of phosphopeptides employed to raise these antibodies are not always provided by companies (including the anti phospho eEF2K antibody validated in this manuscript), making it hard to in-silico predict their cross reactivity before acquisition.Therefore, this manuscript will be highly valuable to prevent researchers interested in studying the regulation of elongation during protein synthesis in C. elegans to go through the laborious validation to detect the inhibitory activity of EEF2 in C. elegans under different cues.The author also provides the conditions and a very neat protocol for the simultaneous detection of phospho EEF2 with a loading control that will prove quite helpful for researchers, saving time and resources.The manuscript is clearly written and easy to read.The experiments are generally carefully performed with appropriate controls.Only minor suggestions and changes should be made (See below) Consider revising or changing the following points (minor suggestions) Inhibitory phosphorylation of eEF2K is elicited in response to different cues.Therefore, it would be really informative to emphasize that the phosphorylation signal observed in figures 2 and 3 is basal.Maybe this could be included within the results section.

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In addition, basal phosphorylation signal is quite high and possibly out of linear range, even with short exposures, which could be troublesome for those aiming to detect subtle differences in phosphorylation among different samples submitted or not to different stress conditions.Perhaps, it would be helpful to add a suggestion in the manuscript for the use of a higher anti-phospho EF2K dilution or alternatively, to make use of a weaker chemiluminescent substrate solution.
○ Conclusion could emphasize the important validation that the load control (tubulin) can be specifically and simultaneously probed in the same membrane.

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The confirmation of the knockout for efk-1KO C. elegans strains is important to prove the specificity of the antibody against EEF2.However, the PCR result (figure 1) was not addressed in the results section, which starts with figure 2. Consider including a simple paragraph with this result in this section.

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Although the author made use of a semi-dry transfer apparatus and membranes are only rinsed in the buffer when this system is employed, it has been previously shown that the composition of buffers may influence the semi-dry transfer efficiency (www.nature.com/protocolexchange/protocols/2925).Therefore, the author could consider including the recipe of the buffer in table 3.

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Maybe I missed the point, but why were membranes first incubated with both antirabbit/anti mouse IgG-HRP and then incubated a second time with anti IgG-HRP against mouse only?(See material and methods section).

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In figure 3B, substitute short for long ○ In the sentences: C. elegans processes eEF2 and eEF2K orthologues = substitute processes for possess.

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After washes, membrane was the incubated with secondary" = substitute the for then.
○ Used as a negative control of efk-1 activity = Change efk-1 for EF2K (the activity refers to the enzyme not the gene itself).

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Antibody for 40 minutes at room temperature, subject to new washes and incubated = substitute subject for subjected.
○ Can be circumvented adding = substitute with: can be circumvented by adding ○ Membrane using semi-dry transfer system substitute with: membrane using a semi-dry transfer system ○ Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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Figure 1 .
Figure 1.efk-1 knockout confirmation by PCR.An efk-1 fragment (~790 bp) was amplified by PCR using specific primer pair to verify the knockout background (efk-1 KO) using N2 worms as positive control and act-1 amplification as load control.

Figure 3 .
Figure 3. Phospho-eEF2 antibody and α-tubulin antibodies can be used simultaneously to C. elegans target detection.Western blot showing that phospho-eEF2 and α-tubulin antibodies can be incubated and developed simultaneously.efk-1 knockout worms were used as negative control to phospho-EEF-2 detection showed in wild-type worms (N2).A) Western blot short exposure (1 min).B) Western blot short exposure (3 min).Indicated molecular weight based on Precision Plus Protein™ Dual Color Standard (Bio-Rad).