Gb11409

The cells were lysed in RIPA lysis buffer (P0013C, Beyotime Biotech., China) containing proteinase and phosphatase inhibitor cocktails. The protein concentration was determined using a BCA protein assay kit (P0009, Beyotime Biotech., China). Equal amounts of total protein were separated in 8%–15% SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked using 5% bovine serum albumin, washed, incubated with primary antibodies at 4 °C overnight, washed, and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibody as appropriate. The protein bands were visualized using enhanced chemiluminescence assay (Thermo Fisher, USA) and exposed to X-ray films. Antibodies for caveolin-1 (GB11409, Servicebio, China), phospho-Akt (T40067S, Abmart, China), Akt (T55561S, Abmart, China), phospho-PI3K(bs-5570R, Bioss, China), PI3K(bs-2067R, Bioss, China), PAI-1(49536S, Cell Signaling Tech., USA), Vimentin (49536S, Cell Signaling Tech., USA), E-Cadherin (14472S, Cell Signaling Tech., USA), N-Cadherin (13116S, Cell Signaling Tech., USA), α-SMA (GB111364, Servicebio, China) were used in western blotting analyses. And β-actin (GB11409, Servicebio, China) was used to confirm equal protein loading.

Liver specimens from rat or mouse were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into sections. The sections were then deparaffinized, hydrated and stained with hematoxylin–eosin (H&E) (Beyotime, Shanghai, China) and Trichrome (Masson) stain kit (Baso diagnostics, Zhuhai, China). Hepatic lipid content was quantified on fresh frozen sections using Oil Red O staining (Beyotime, Shanghai, China). For immunohistochemistry, the liver sections are incubated in 3% H₂O₂ for 25 min following antigen retrieval, and then blocked with 3%BSA for 30 min. After that, the sections were incubated with the following primary antibody at 4 °C overnight: mouse anti-CAV2 (ab2911; Abcam, Cambridge, UK), mouse anti-CAV1 (GB11409; Servicebio; Wuhan; China), mouse anti-collagen1α1 (GB11022-3; Servicebio; Wuhan; China), rabbit anti-CTGF (ab6992; Abcam, Cambridge, UK), rabbit anti-TGFβRI (sc-398; Santa Cruz Biotechnology, CA, USA), rabbit anti-α-Dynamin2 (ab151555; Abcam, Cambridge, UK), and then with a biotinylated secondary antibody for 50 min. The expression was visualized by 3,30-diaminobenzi-dine tetrahydrochloride (DAB) staining. And the immunohistochemistry is quantified by Image J software.