{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T16:24:56Z","timestamp":1791822296116,"version":"4.3.4"},"reference-count":23,"publisher":"Oxford University Press (OUP)","issue":"2","license":[{"start":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T00:00:00Z","timestamp":1765324800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"name":"National Heart, Lung, and Blood Institute of the National Institutes of Health","award":["NIH R01HL122546"],"award-info":[{"award-number":["NIH R01HL122546"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,12,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Protein O-GlcNAcylation is a dynamic post-translational modification with emerging roles in cardiac pathophysiology. The availability of different pan-specific antibodies to assess global O-GlcNAc levels, and variability in western blot results has hindered cross-study reproducibility and interpretation. In this study, we applied optimized immunoblotting protocols using both CTD110.6 and RL2 O-GlcNAc antibodies, alongside subcellular fractionation, to investigate temporal and sex-specific changes in cardiac O-GlcNAcylation during pressure overload hypertrophy (POH) from transverse aortic constriction (TAC) during early (1-week POH, 1wTAC) and chronic (6-weeks POH, 6wTAC) POH in mice. Global O-GlcNAc levels were elevated in early POH and returned to baseline in chronic POH, consistent across both antibodies and sexes. Subcellular fractionation revealed persistent O-GlcNAc elevations in cytoplasmic and membrane fractions in chronic POH for both sexes, which were not detected in unfractionated samples. Female mice exhibited significantly higher O-GlcNAc levels than males during POH, particularly at early POH, highlighting sex-specific regulation. OGT and OGA protein levels also varied by compartment and sex, suggesting differential enzymatic control. In conclusion, our findings underscore the importance of methodological rigor in O-GlcNAc detection and demonstrate that fractionation enhances sensitivity to subtle changes in cardiac O-GlcNAcylation. Our principal new findings are protein O-GlcNAcylation dysregulation continues from early POH (1wTAC) into chronic POH (6wTAC groups) along with showing differences in O-GlcNAc levels between males and females during POH. These results provide new insights into the temporal and sex-dependent dynamics of O-GlcNAc signaling in POH and support its potential as a therapeutic target in cardiovascular disease.<\/jats:p>","DOI":"10.1093\/glycob\/cwaf086","type":"journal-article","created":{"date-parts":[[2025,12,10]],"date-time":"2025-12-10T13:08:17Z","timestamp":1765372097000},"source":"Crossref","is-referenced-by-count":2,"title":["Detection of cardiac O-GlcNAcylation via subcellular fractionation and dual antibody analysis in pressure overload cardiac hypertrophy"],"prefix":"10.1093","volume":"36","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1041-4795","authenticated-orcid":false,"given":"Dolena","family":"Ledee","sequence":"first","affiliation":[{"name":"Norcliffe Foundation Center for Integrative Brain Research, Seattle Children\u2019s Research Institute , 1900 9th Ave., Seattle, Washington 98101,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei Zhong","family":"Zhu","sequence":"additional","affiliation":[{"name":"Norcliffe Foundation Center for Integrative Brain Research, Seattle Children\u2019s Research Institute , 1900 9th Ave., Seattle, Washington 98101,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron K","family":"Olson","sequence":"additional","affiliation":[{"name":"Norcliffe Foundation Center for Integrative Brain Research, Seattle Children\u2019s Research Institute , 1900 9th Ave., Seattle, Washington 98101,","place":["United States"]},{"name":"Division of Cardiology, Department of Pediatrics, University of Washington , 1959 NE Pacific St, Seattle, Washington 98101,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2025,12,10]]},"reference":[{"key":"2026012614270961500_ref1","doi-asserted-by":"publisher","first-page":"440","DOI":"10.1016\/j.mex.2015.11.001","article-title":"Sequential 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