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Analysis of institutional authors

Ripa, InesAuthorBello-Morales, RaquelCorresponding AuthorLopez-Guerrero, Jose AntonioCorresponding Author

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August 4, 2025
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Polyclonal LC3B Antibodies Generate Non-Specific Staining in the Nucleus of Herpes Simplex Virus Type 1-Infected Cells: Caution in the Interpretation of LC3 Staining in the Immunofluorescence Analysis of Viral Infections

Publicated to: International Journal Of Molecular Sciences. 26 (14): 6682- - 2025-07-11 26(14), DOI: 10.3390/ijms26146682

Authors:

Ripa, I; Andreu, S; Galdo, D; Caballero, O; Bello-Morales, R; López-Guerrero, JA
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Affiliations

CSIC, Ctr Biol Mol Severo Ochoa, Madrid 28049, Spain - Author
Univ Autonoma Madrid, Dept Mol Biol, Madrid 28049, Spain - Author

Abstract

The most common marker used to monitor autophagy is the microtubule-associated protein light chain 3 (LC3). Upon induction of autophagy, LC3 is conjugated to phosphatidylethanolamine and targeted to autophagic membranes, which can be easily detected by immunofluorescence. However, this technique has some limitations. During the early stages of HSV-1 infection, strong LC3B nuclear staining is observed within the viral replication compartments. This staining is only detected when using polyclonal antibodies. It is noteworthy that monoclonal antibodies or the GFP-LC3 plasmid do not reveal any nuclear LC3 staining. Interestingly, LC3B is not detected in the nuclear fraction of infected cells by Western blotting, even when polyclonal antibodies are used. In infected LC3B knockout cells, nuclear staining is still observed when using polyclonal LC3B antibodies. This suggests that polyclonal LC3B antibodies generate non-specific nuclear staining in infected cells, which could result in misinterpretation and erroneous conclusions. These findings raise questions about the reliability of LC3-immunofluorescence assays in herpesvirus infections. It is imperative that the methodology employed for monitoring autophagy by immunofluorescence in viral infections be reviewed and updated, and that the specificity of anti-LC3B antibodies be tested before use. To ensure the accuracy of the results, it is essential to validate this technique with additional assays, such as by immunoblot analysis or via the use of autophagy-deficient cell lines.
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Keywords

ActivationAnimalsAntibodiesAutophagyCell nucleusChlorocebus aethiopsCompartmentsDegradationFluorescent antibody techniqueForHerpes simplexHerpes simplex virus type 1Herpesvirus 1, humanHumansInhibitionLc3 antibodyMap1lc3b protein, humanMicrotubule-associated proteinsNeurovirulenceOligodendrocytOligodendrocytePkrProteinsPunctaReplicationReplication compartmentStaining and labeling

Quality index

Bibliometric impact. Analysis of the contribution and dissemination channel

The work has been published in the journal International Journal Of Molecular Sciences due to its progression and the good impact it has achieved in recent years, according to the agency WoS (JCR), it has become a reference in its field. In the year of publication of the work, 2025, it was in position 72/320, thus managing to position itself as a Q1 (Primer Cuartil), in the category Biochemistry & Molecular Biology.

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Impact and social visibility

From the perspective of influence or social adoption, and based on metrics associated with mentions and interactions provided by agencies specializing in calculating the so-called "Alternative or Social Metrics," we can highlight as of 2026-01-15:

  • The use, from an academic perspective evidenced by the Altmetric agency indicator referring to aggregations made by the personal bibliographic manager Mendeley, gives us a total of: 3.
  • The use of this contribution in bookmarks, code forks, additions to favorite lists for recurrent reading, as well as general views, indicates that someone is using the publication as a basis for their current work. This may be a notable indicator of future more formal and academic citations. This claim is supported by the result of the "Capture" indicator, which yields a total of: 3 (PlumX).

With a more dissemination-oriented intent and targeting more general audiences, we can observe other more global scores such as:

  • The Total Score from Altmetric: 2.
  • The number of mentions on the social network X (formerly Twitter): 2 (Altmetric).

It is essential to present evidence supporting full alignment with institutional principles and guidelines on Open Science and the Conservation and Dissemination of Intellectual Heritage. A clear example of this is:

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Leadership analysis of institutional authors

There is a significant leadership presence as some of the institution’s authors appear as the first or last signer, detailed as follows: First Author (RIPA PERALTA, INES) and Last Author (LOPEZ GUERRERO, JOSE ANTONIO).

the authors responsible for correspondence tasks have been BELLO-MORALES ARROYO, ANGELES RAQUEL and LOPEZ GUERRERO, JOSE ANTONIO.

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Awards linked to the item

This research was funded by the Ministerio de Ciencia e Innovacion, Spain (PID2022-140632NB-I00).
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