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. 2017 Jul 13;1(7):675–690. doi: 10.1002/hep4.1066

Figure 7.

Figure 7

Secretion of elevated EV CYP2E1 by alcohol exposure is dependent on oxidative and ER stress. (A) Immunoblot analyses of CYP2E1, PDI, and Grp78 in hepatocyte lysates after primary hepatocytes were treated with 100 mM EtOH in the absence or presence of 1 mM NAC, 40 μM CMZ, 5 μM 4‐PBA, or 1 μM THAP. (B) Total number of EVs in each indicated group was measured by Nanosight. (C) Immunoblot analyses of CYP2E1, PDI, and Grp78 in hepatocyte lysates after primary hepatocytes were treated with 100 mM EtOH with or without 20 μM Cyto‐D. β‐Actin, used as a loading control, is shown. (D) Total number of EVs in the each indicated group was measured by Nanosight. (E) EV proteins from the indicated groups were immunoprecipitated with anti‐CYP2E1 antibody. The immunoprecipitated proteins were then subjected to immunoblot analysis with anti‐3‐NT or anti‐CYP2E1 antibody. The average ratio of 3‐NT/CYP2E1 for each group is shown at the bottom. (F) CYP2E1 activity was measured by hydroxylation of p‐nitrophenol and expressed as pmol/min/mg of protein. **P < 0.01. Abbreviations: 3‐NT, 3‐nitrotyrosine; Con, control; EtOH, ethanol; NAC, N‐acetylcysteine; 4‐PBA, 4‐phenylbutyric acid; THAP, thapsigargin. Data are shown as means ± SD.