Peer-reviewed by 2 reviewers with median rating of 12.5/20. Review process was triple-blinded.
Round 1 (11.5/20)
Round 2 (12.5/20)
Conceptual advance and Impact8
The manuscript focused on studying the expression of endogenous halfAgo2 protein form . The author used different complementary approaches that provided several evidences which strongly supports the conclusions drawn by the authors.
I recommend accepting the manuscript as it is.
Conceptual advance and Impact5
The possibility of a shorter Ago2 form is an interesting idea. However, the observations are weak due to absence of solid evidence to prove its existence (they could be degradation products due to improper handling of the lysate) and its functional implications.
Please modify according to the review discussed below.
Blots are cropped too close. Please provide complete blot.
Remove data showing effect of miRNA on Ago2/halfAgo2 (F)
Please include data for other proteins (as discussed below).
Please include data for primary cell lines (as discussed below).
Results & Discussion
1. Can the authors prove that halfAgo2 is not a product of degradation of proteins due to improper handling of the cellular lysate? What about other proteins in the same lysate? Do they also produce such shorter isoforms?
2. Can both Ago2 and halfAgo2 be expressed (protein expression and purification) in bacteria?
3. MCF7, T47D, U2OS and HeLa are all cancer cell lines? Have the authors tested primary cell lines?
4. The quantification of the blot showing the effect of miRNA on Ago2/halfAgo2 is not correct. The first lane is clearly overloaded (based on actin) and thus, has less protein compared to the last lane. Please provide blots from all replicates with proper quantitation. Alternatively, this data must be deleted.
5. The ratios of Ago2/halfAgo2 are different in B (Upper panel) and D. From the results, it appears that the addition of FLAG stabilizes the full length protein. Can the authors provide an explanation for this?
6. More biological replicates must be tested (particularly in A) and the results must be provided in supplementary info.
This limitation must be addressed. What technical limitations prevent the isolation of both forms?