The approach of this paper, i.e. extensive evaluation of ERK-MAPK signalling factors by immunofluorescence labelling of human skin, is highly informative and helpful for the research field. I believe this paper fits the concepts of GigaScience and deserves acceptance in the journal. Several revisions, however, should be considered by the authors before final acceptance.

Major Compulsory Revisions:

1. I don't think the authors' conclusion in abstract, "These data indicate sustained ERKMAPK signalling during the two weeks of keratinocyte differentiation" is supported by their experiments. Certainly the human skin shows similar ERK activity across the depth of the skin (Figure AF3.10 and AF3.11), but the authors cannot exclude the possibility that this is due to low sensitivity of ERK activity in the system, probably due to lack of positive control that shows high ERK activity (e.g. human skin sample from patients with psoriasis or ). Even if the ERK activity is spatially homogeneous, ERK activity may reveal dynamic temporal changes during turnover, which cannot be described as "sustained". The authors should avoid mentioning temporal changes in the conclusion and instead, should describe spatial ERK-MAPK signalling activity in the different layers.

2. In Figure AF3.10 and AF3.11, the authors should show cytoplasm-to-nucleus ratio of ERK fluorescence, which will help the understanding and interpretation of the authors' discussion below the Figure AF3. 11. Although the authors write "it has been suggested that the accumulation of ERK-MAPK components is independent of nucleocytoplasmic shuttling rates", the paper they cited (54) reads "varying the nucleocytoplasmic shuttling velocities of (phosphor-)ERK significantly varied the level of phosphor-ERK in the nucleus. Thus the maximum level of phosphor-ERK accumulation in the nucleus seems to be dependent primarily on the nucleocytoplasmic shuttling velocities of ERK.". The authors should explain this contradiction and, if necessary, they should correct their discussion.

Major Discretionary Revisions:

1. In Table1, the authors listed image data quality using black and white circles. This is a good way to help intuitive understanding, but the authors should mention how they determined the quality (e.g., consistency with reported results, signal to noise ratio, mean signal intensity, efficiency in cell segmentation etc.). If quality was determined in a subjective manner, it should be clarified in the text.

2. In Figure AF3.6 B, I see strong fluorescence in nuclei of cells in suprabasal layer, which is an unexpected result since Raf is basically localized in cytoplasm and membrane. Authors should show their interpretation of this in the text (e.g., non-specific binding of the primary antibody or the actual nuclear localisation of Raf).

Minor Revisions: The reference in Additional File1 includes "!!!INVALID CITATION!!!" (9).

Level of interest Please indicate how interesting you found the manuscript: An article of importance in its field

Quality of written English
Please indicate the quality of language in the manuscript: Acceptable

Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:

1. Have you in the past five years received reimbursements, fees, funding, or salary from an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future?

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6. Do you have any non-financial competing interests in relation to this paper? If you can answer no to all of the above, write 'I declare that I have no competing interests' below. If your reply is yes to any, please give details below.

I declare that I have no competing interests.

I agree to the open peer review policy of the journal. I understand that my name will be included on my report to the authors and, if the manuscript is accepted for publication, my named report including any attachments I upload will be posted on the website along with the authors' responses. I agree for my report to be made available under an Open Access Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any comments which I do not wish to be included in my named report can be included as confidential comments to the editors, which will not be published.

I agree to the open peer review policy of the journal.

The reviewed version of the manuscript can be seen here:

All revised versions are also available:

The authors adequately addressed my previous concerns. I am happy to recommend this paper for publication after the following very minor revision.

Minor Revisions: I still see some errors in references of AF2 and AF4: !!! INVALID CITATION !!! and unreadable letters (�).

Level of interest
Please indicate how interesting you found the manuscript: An article of importance in its field

Quality of written English
Please indicate the quality of language in the manuscript: Acceptable

Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:

1. Have you in the past five years received reimbursements, fees, funding, or salary from an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future?

2. Do you hold any stocks or shares in an organisation that may in any way gain or lose financially from the publication of this manuscript, either now or in the future?

3. Do you hold or are you currently applying for any patents relating to the content of the manuscript?

4. Have you received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript?

5. Do you have any other financial competing interests?

6. Do you have any non-financial competing interests in relation to this paper?

If you can answer no to all of the above, write 'I declare that I have no competing interests' below. If your reply is yes to any, please give details below.

I declare that I have no competing interests.

I agree to the open peer review policy of the journal. I understand that my name will be included on my report to the authors and, if the manuscript is accepted for publication, my named report including any attachments I upload will be posted on the website along with the authors' responses. I agree for my report to be made available under an Open Access Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0/). I understand that any comments which I do not wish to be included in my named report can be included as confidential comments to the editors, which will not be published.

I agree to the open peer review policy of the journal.

The reviewed version of the manuscript can be seen here:

All revised versions are also available: