Western blot analysis

Cells were lysed with cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail, 1% PMSF, and 1% phosphatase inhibitor. Protein concentrations were measured by the BCATM Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) using BSA as standard. Appropriate amount of protein samples (40 μg for liver tissues; 25–50 μg for cells) along with 4× loading buffer and ddH₂O were boiled for 4 min and then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Following by electrophoresis, the separated proteins were blotted onto polyvinylidene fluoride (PVDF) membranes in transfer buffer with constant current of 300 mA for 3 h at 4 °C. Then the PVDF membranes were sequentially washed with TBST containing 0.2% Tween-20, blocked with 5% nonfat milk in TBST and incubated with the interested primary antibodies diluted in TBST containing 0.2% Tween-20 overnight at 4 °C. Antibodies used for immunoblotting included VEGFR2 (1:1000, rabbit monoclonal, Cell Signaling Technology, #9698), eNOS (1:1000, mouse monoclonal, Abcam, ab76198), VE-Cadherin (1:1000, rabbit polyclonal, Abcam, ab33168), PCNA (1:1000, rabbit monoclonal, Cell Signaling Technology, #13110), α-SMA (1:1000, rabbit polyclonal, Abcam, ab5694), COL1α1 (1:1000, rabbit polyclonal, Abcam, ab34710), MMP2 (1:1000, rabbit monoclonal, Abcam, ab92536), TIMP1 (1:1000, mouse monoclonal, Santa Cruz, sc-21734), KLF2 (1:1000, mouse polyclonal, Santa Cruz, sc-166238). GAPDH were severed as control for total protein amount. Then, all of membranes were incubated with the HRP-conjugated secondary antibody for 1 h at room temperature. Every experiment was repeated at least three times independently.