Ab151555

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and the cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated in PBS with non-fat milk and probed with an anti-CHC antibody (1:1,000; ab21679, Abcam), anti-dynamin II antibody (1:1,000; ab151555, Abcam), anti-caveolin-1 antibody (1:1,000; C4490, Sigma), anti-Rab5 antibody (1:1,000; ab18211, Abcam), or an anti-Rab7 antibody (1:1,000; ab126712, Abcam) for 1 h at room temperature and subsequently washed three times with 0.1% Tween 20/PBS, followed by an incubation for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:10,000; BA1054, Boster) or HRP-conjugated goat anti-mouse secondary antibody (1:10,000; BA1050, Boster). The membranes were washed three times with 0.1% Tween 20/PBS and visualized by exposure to FluorChem HD2 Imaging System (Alpha Innotech) after the addition of a chemiluminescent substrate (SuperSignal West Dura Extended Duration Substrate; 34075; Thermo Scientific Pierce) (Li M. et al., 2017 (link)).

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.

Bortezomib and endocytosis inhibitors, including heparin, chlorpromazine, amiloride, dynasore, wortmannin, omeprazole, and genistein were purchased from Selleck Chemicals (Houston, TX, USA). 5-(N-Ethyl-Nisopropyl) amiloride (EIPA) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against flotillin-1 (Flot1, D2V7J, 18634T, 1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, D16H11, 5174S, 1:1000), calreticulin (D3E6, 12238T, 1:1000), caveolin-1 (CAV-1, D46G3, 3267T, 1:1000), and clathrin heavy chain (CLTC, D3C6, 4796S, 1:1000) were purchased from (Cell Signaling Technology, Danvers, MA, USA). Antibodies against dynamin-2 (DNM2, EPR9053, ab151555, 1:1000) and CD9 (EPR2949, ab92726, 1:2000) were bought from Abcam (Cambridge, United Kingdom). Anti-human CD63 antibody (Ts63, 10628D, 1:250) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). IRDye 680RD or 800CW goat anti-mouse/rabbit IgG secondary antibodies (1:10000) were purchased from LI-COR Biosciences (Lincoln, NE, USA).