Western blotting
T-PER (Thermo Fisher Scientific Inc.) and Halt protease and phosphatase
inhibitor cocktail (Thermo Fisher Scientific Inc.) were mixed at a ratio
of 99:1 to prepare the lysis buffer. Liver samples were homogenized in
the lysis buffer and centrifuged at 10,000 × g for 10 min. The
supernatants were collected, each sample was mixed with Lane marker
reducing sample buffer (Thermo Fisher Scientific Inc.) at a ratio of
4:1, and heated at 95 ºC for 5 min to prepare the blotting samples. The
prepared samples were separated using sodium dodecyl
sulphate-polyacrylamide gel electrophoresis. Proteins were transferred
from the gel onto a PVDF membrane using an iBlot Dry Blotting System
(Thermo Fisher Scientific Inc.). The primary antibodies reactions
[anti-Vinculin antibody (Cat# 13901S, RRID:AB_2728768) was purchased
from Cell Signaling Tech, Inc.; anti- NQO-1 antibody (Cat# ab34173,
RRID:AB_2251526), anti-Glutamate-cysteine ligase catalytic subunit
(GCLC) antibody (Cat# ab207777, RRID: none yet assigned), and
anti-alpha smooth muscle actin (αSMA) antibody (Cat# ab32575,
RRID:AB_722538) were purchased from Abcam Inc. (Cambridge, UK) ] were
performed using the iBindTM Western Device (Thermo
Fisher Scientific Inc.). The bound primary antibodies were detected
using an HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat #
HAF008, RRID: AB_357235; R&D Systems Inc., Minneapolis, MN). Then,
AmershanTM ECL Prime western blotting detection
reagent (GE Healthcare Inc., Chicago, IL) and
ChemiDocTM XRS Plus (Bio-Rad Laboratories Inc.,
Hercules, CA) were used for protein detection. All immunological
procedures were performed according to the recommendations of theBritish Journal of Pharmacology (Alexander et al., 2018).