Western blot
For Western blot, the total protein were extracted from the cells by
homogenization in RIPA lysis buffer supplemented with 1 mmol PMSF
(Pierce, Rockford, IL). Upon centrifugation at 14,000g for 10 min
at 4 oC, the supernatant was collected, and the total
protein was quantified using the BCA protein assay kit (Beyotime,
Shanghai) following the manufacturer instructions. The protein was
separated by 10% SDS-polyacrylamide electrophoresis gels and
electrotransferred onto a PVDF membrane (Millipore, Beijing). After
blocking with 5% nonfat dry milk in TBST, the membrane was incubated
with mouse anti-α-SMA, rabbit
anti-MMP-2, rabbit anti-MMP-9, rabbit anti-collagen I, rabbit
anti-collagen III (1:1000, Abcam), rabbit NF-κB p-P65 (1:500), NF-κB P65
(1:500),p-IκBα (1:500), IκBα (1:500), and PPAR-γ (1:500) (1:1000, Cell
Signaling, Danvers, MA ) and mouse anti-β-actin (1:2000, Abcam)
overnight at 4 oC. Then, the membrane was washed with
TBST and then incubated with goat anti-rabbit or goat anti-mouse
(1:2000, santa cruz, CA). The proteins were visualized using enhanced
chemiluminescence (Bio-Rad, Hercules, CA).