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. 2023 Jan 27;13:1079962. doi: 10.3389/fimmu.2022.1079962

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Copyright © 2023 Gandhirajan, Roychowdhury, Kibler, Cross, Abraham, Bellar, Nagy, Scheraga and Vachharajani

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The effect of acute ethanol-exposure on pAtg4B and microtubule associated protein 1 light chain-3B (LC3) I and II expression in macrophages. (A) WT-BMDM were exposed to vehicle or ethanol ± LPS. Atg4B was immunoprecipitated (IP) from whole-cell lysates of WT-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (B) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (C) SIRT2KO-BMDM were exposed to ethanol ± LPS. IP for Atg4B from whole-cell lysates of SIRT2KO-BMDM using an anti-Atg4B antibody followed by IB analysis of pSerine and total Atg4B. IP with IgG control antibody was used as a negative control. (D) Western blot analysis of total Atg4B in the whole cell lysate used as input for the Atg4B IP. (E) LC3-I and LC3-II expression were analyzed by western blot in WT-BMDM exposed to vehicle or ethanol ± LPS. Western blot image quantification of ethanol vs. vehicle-expose WT-BMDM± LPS, showing LC3-I in (F) and LC3-II in (G), normalized to vehicle control (Vehicle-LPS) (n = 4 blots; * p < 0.05). (H) Western blot analysis of LC3-I and II expression in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS. Western blot image quantification in Ethanol-exposed WT-BMDM and SIRT2KO-BMDM ± LPS for LC3-I in (I) and LC3-II in (J), normalized to ethanol control (Ethanol-LPS) (n = 4 blots; * p < 0.05). (K). Western blot analysis of LC3-I and II expression of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS. Western blot image quantification of Ethanol-exposed WT-BMDM treated with AK-7 or DMSO ± LPS for LC3-I in (L) and LC3-II in (M) normalized to ethanol control (Ethanol-LPS) (n = 3 blots; * p < 0.05).