Proteomic analysis of circulating EVs isolated from APAP-exposed mice and confirmation of various proteins by immunoblot analysis.

<p>(A) Proteomic analysis of EV proteins isolated from Control or APAP-exposed mice using 2D LC-MS/MS. Differentially expressed proteins were identified by bioinformatic analysis, as described. (B) Classification based on the subcellular location of the identified proteins. Comparison of proteomic data from the EVs in this study with those of rat primary hepatocytes, as reported previously by our group[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172463#pone.0172463.ref030" target="_blank">30</a>]. (C) The 380 proteins in EVs from control plasma and 431 proteins in EVs from APAP plasma were classified by tissuespecific origin and categorized according to the DAVID program. Some proteins are classified to more than two organs. (D) Comparison of differentially expressed proteins in circulating EVs from APAP-treated groups with the previously reported results[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172463#pone.0172463.ref039" target="_blank">39</a>] from hepatocytes treated with APAP, based on biological process analysis. (E) Confirmation of liver-specific proteins, inflammation-related proteins, and exosomal marker proteins in circulating EVs by immunoblot analysis, as indicated. (F) Protein levels of ASS1 and CES1 were measured in whole liver lysates from the indicated groups.