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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1:  The manuscript authored by Yadav et al.., entitled “Rapid purification of brain protein complexes containing active and inactive forms of the G protein Gαo” presents a study aimed at identifying downstream effectors of the Gαo protein, the most abundant heterotrimeric G protein in the brain, by developing methods to isolate native Gαo-GTP protein complexes from mouse brain lysates. The authors employ immunoprecipitation and size exclusion chromatography to analyze Gαo complexes, demonstrating changes in complex size upon activation with GTPγS. The study addresses a significant gap in understanding Gαo signaling, as no direct effectors have been well-characterized despite its abundance and role in inhibiting neural function. The methods are novel and well-executed, but several areas require clarification, additional controls, and improved discussion to strengthen the manuscript's impact and clarity.

My comments are as follows:

1. The introduction mentions genetic and interaction screens that failed to identify Gαo effectors, but does not compare the current method’s advantages over these approaches in detail. A stronger rationale for why biochemical purification will likely succeed where genetic screens failed would strengthen the manuscript. Expand the introduction to discuss the limitations of yeast two-hybrid and genetic screens more explicitly and highlight how the current method overcomes these.

2. The manuscript aims to identify Gαo effectors but does not report any specific effector proteins identified through the described methods. The discussion mentions that effectors may be present at lower abundance and detectable by mass spectrometry, but no such data are provided. This is a significant limitation, as the study's primary goal appears unfulfilled. If mass spectrometry was performed, include preliminary results or clarify why such data are not presented. If not performed, explicitly state this as a limitation and outline plans for future work to identify effectors using these methods.

3. The observed molecular weights of Gαo complexes (1249–945 kDa for GDP-bound and 98–398 kDa for GTPγS-bound) are much larger than expected, even accounting for Triton X-100 micelles. The manuscript attributes this to potential effector interactions but does not adequately discuss alternative explanations, such as non-specific protein aggregation or micelle size variability. Include controls to rule out non-specific interactions, such as comparing Gαo complexes to those of other G proteins (e.g., Gαq or Gαi) under identical conditions. Discuss the impact of detergent micelles on apparent molecular weight more thoroughly, potentially referencing studies on membrane protein complexes in similar systems.

4. Validate anti-Gαo antibody specificity by testing it against lysates from Ga0 knockout mice or performing co-immunoprecipitation with other G protein subunits (e.g., Gαq or Gαi) to confirm selectivity.

5. The manuscript does not mention the number of biological or technical replicates for key experiments (e.g., immunoprecipitation, size exclusion chromatography). The statement “we prepared brain lysates multiple times” is vague. Specify the number of replicates and include statistical analysis where applicable (e.g., for protein yield or band intensity in western blots). Provide error bars or variability measures for quantitative data.

6. Figures 1B and 1C are referenced extensively, but the manuscript does not clearly describe the expected molecular weights of Gβ and G bands. The text mentions Gβ at 37 kDa and Ga0 at 40 kDa, but G is not quantified. Annotate figures with molecular weight markers and clarify G band identification in the text.

7. The term “GTPyS” is used in the abstract and early sections, while “GTP S” is used later. This inconsistency may confuse readers. Standardize to “GTP S” throughout the manuscript.

8. The manuscript presents a technically sound and novel approach to studying Gαo signaling by isolating native Gαo-GTP complexes from mouse brain lysates. The methods are well-executed and have the potential to advance the field, particularly if paired with downstream analyses like mass spectrometry. However, the lack of effector identification, incomplete validation of antibody specificity, and limited discussion of negative results weaken the current version. Addressing the major concerns (particularly providing effector data or clarifying its absence) and incorporating the minor suggestions will significantly strengthen the manuscript. I recommend major revisions before publication, focusing on clarifying results, adding controls, and enhancing the discussion.

Reviewer #2:  The authors have done a beautiful job of purifying complicated G�o protein from mouse brain lysate and tried to identify its binding to effectors in the manuscript. They have given a detailed introduction to the protein of interest and a detailed methodology of their experiments. Though the study in the manuscript is incomplete and inconclusive, it forms a base for future studies with these proteins.

I found the manuscript interesting, and it also projects interesting work. Having said that, there are some concerns about the manuscript, which would enhance the manuscript and help in future research directions even better. Below are my comments on the manuscript:

1. In the introduction part, it would help to provide some information about the effectors of G�o proteins. The authors have abruptly jumped into discussing effector proteins.

2. The authors have mentioned SNARE proteins. Nowhere in the manuscript have they described the abbreviation and its significance.

3. The authors say they prepared brain lysates multiple times to get 12 mg/ml. The scientific way of saying this would be to quantitate it by saying how many brain samples.

4. There are a couple of abbreviations for IP. The authors should either use the full form for one of them or should name the buffer with some other acronym.

5. The manuscript in general has errors/typos that need to be corrected. Also, the units should be written in a consistent manner throughout the manuscript like �L, mM, �M etc.

6. For SEC, the authors have mentioned the specific fraction groups collected but never mentioned the rationale of it. The better way of saying this would be all the fractions were collected.

7. The authors have mentioned GTP�S as a non-hydrolysable analog of GTP. However, it is a slowly hydrolyzing analog. For this study, better suited would have been other non-hydrolysable analogs such as GMPPCP/GMPPNP.

8. An alternative method to check the loading could have been HPLC, which would be more quantitative and unbiased.

9. In the western blot of fig. 1, the loading controls are absent, which is standard for the blot images.

10. In the blot of fig 1C, the G�o bands should be identical, as per the hypothesis and explanations in the text. Nothing has been discussed why do you see a difference in intensity of G�o in the GDP and GTP�S blots.

11. The author say mass spectrometry could be used to check the effector-binding. A lane in the SDS-PAGE with crude extract, which is not modified to load any nucleotide, would be an interesting sample to compare.

12. In fig.2, it is interesting and confusing at the same time, as to how there is a huge fraction from 37-45 which absorbs at 280, but does not read in Coomassie. It would be of interest to get the MALDI done for these samples and check the possibility. That may also give a conclusion to the manuscript.

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Reviewer #1: No

Reviewer #2: No

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Rapid purification of brain protein complexes containing active and inactive forms of the G protein Gαo

PONE-D-25-19360R1

Dear Dr. Kumar,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Ajit Prakash, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: N/A

Reviewer #3: (No Response)

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: No

Reviewer #3: No

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