Anti il 21

Total proteins were extracted from cells and tissues. The cells and tissues were lysed with 20 μL of RIPA Buffer (Roche Molecular Biochemicals, Switzerland), and protein concentrations were detected using the BCA kit (Beyotime, China), according to the manufacturer’s guidelines. Protein samples (20~40 μg) were separated on an SDS-PAGE gel and transferred to nitrocellulose membranes. After blocking the membranes with 5% skim milk in phosphate-buffered saline (PBS) for 1 h at room temperature, the membranes were probed with anti-IL-21 (Abcam, UK), anti-claudin-5 (GeneTex, USA), anti-p-NF-κB (Santa Cruz Biotechnology, USA), anti-NF-κB (Santa Cruz Biotechnology, USA), anti-p-ERK1/2 (Proteintech, USA), anti-ERK1/2 (Proteintech, USA), anti-p-JNK (Santa Cruz Biotechnology, USA), anti-JNK (Proteintech, USA), anti-p-P38 (Santa Cruz Biotechnology, USA), anti-P38 (GeneTex, USA) and GAPDH (Zhongshanjinqiao, China) antibodies at 4°C overnight. The nitrocellulose membranes were washed with PBST (PBS containing 0.5% Tween 20) three times for 7 min each, which was followed by the incubation with a fluorescence-labeled secondary antibody (IRDye700/800 mouse and rabbit antibodies) (Santa Cruz Biotechnology, USA). Protein levels were determined using an Odyssey infrared scanning system (LI-COR Biosciences, USA), and the band intensities were quantified using ImageJ software.

Mice were sacrificed and liver tissues were harvested at 0, 4, 7, 9, 12, 16 weeks and fixed in 10% neutral buffered formalin. Liver sections (4 μm) were treated with 0.3% H2O2 and 10% normal goat serum (Boster, Wuhan, China) to block endogenous peroxidases and non-specific binding. The slides were incubated with anti-alpha smooth muscle actin (Abcam) and anti-IL-21 (Abcam) for 1 hours and further incubated with the secondary antibodies (ChemMate Envision⁺/HRP/DAB, rabbit/mouse detection kit, Gene-tech, Shanghai, China). Six random digital images of each mouse were captured using a light microscope (OLYMPUS) and the dark brown were positively staining cells which were analyzed with Image-Pro Plus 6.0 software.