Horseradish peroxidase conjugated secondary antibody

The regenerating liver tissues stored in liquid nitrogen were ground into fine
powder and then suspended in extraction buffer (7 M urea, 2 M thiourea, 4%
CHAPS). Next, the suspension was vortex-mixed for 1 h at 4 °C, and subsequently
centrifuged at 20,000 x g for 1 h at 4 °C. The supernatants
were collected and stored at –80 °C for further use. The protein concentration
was assessed with the commercial RC DC^TM Protein Assay Kit according
to the manufacturer’s instructions (BIO-RAD, USA). Protein samples, 50 μg, were
separated by electrophoresis on 12% SDS/PAGE gels and subsequently
electrophoretically transferred to polyvinylidene difluoride membranes
(Millipore). The membranes were blocked with 5% non-fat milk, washed, and
subsequently probed with antibodies against 14-3-3β/α, γ, ζ/δ, σ, ε, η, τ/θ (all
1:1000) and GAPDH (1:2000) (Sangon Biotech Co. Ltd., Shanghai, China)overnight
at 4 °C. After being washed, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibodies (Sangon Biotech Co. Ltd., Shanghai,
China), detected with an enhanced chemiluminescence detection kit (Boster
Corporation, China) and then imaged in an ImageQuant LAS 4000 mini (GE
Healthcare Bio-Sciences Corporation) system.

Homogenized tissue or harvested cells in a commercial lysis buffer (Beyotime, Zhejiang, China) were first quantified using the BCA method (Pierce; Rockford, IL, USA), and then denatured by boiling for 5 min. Forty micrograms of protein per sample was denatured, separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and 5% (w/v) nonfat dry milk for 1 h. The membranes were then incubated overnight at 4°C with the primary antibodies of α-smooth muscle actin (SMA), SIRT1, p53, acetylated p53 (Ac-p53), collagen I, caspase3, and β-actin (Abcam, Cambridge, MA) at a dilution of 1:1000. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Sangon, China) at a dilution of 1:2000 at room temperature for 1 h. The signals were visualized using an electrochemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA) following the manufacturer’s instructions and captured digitally using chemiluminescence imaging systems (Shanghai Clinx Science Instruments, China). The bands were quantitated using ImageJ software.

HFL1 cells were pretreated with the desired doses of GSPs (2.5, 5.0 or 10 μg/ml) or MitoQ (200 nM) in DMSO for 24 h. Then, the cells were irradiated with 8 Gy and incubated for 1 h or 72 h. Total cellular samples were washed twice with ice-cold PBS and incubated for 10 min in lysis buffer. Protein concentrations were determined using the BCA Protein Assay Kit (Sangon Biotech, Shanghai, China). Equal amounts of sample protein (20 μg) were separated using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk reagent dissolved in 1 × phosphate-buffered saline with 0.05% Tween-20 and then incubated with anti-α-SMA (Abcam, Cambridge, UK), fibronectin, p38MAPK, p-p38MAPK, Akt, p-Akt (Santa Cruz, CA) and GAPDH antibodies (Sangon Biotech, Shanghai, China) at 4 °C for 12 h. Protein bands were detected by incubating with horseradish peroxidase-conjugated secondary antibodies (Sangon Biotech, Shanghai, China), which were visualized with enhanced chemiluminescence reagent (Beyotime, Jiangsu, China).