Ab71395

For the western blot analyses, RIPA buffer containing protease inhibitors and phospha tase inhibitors (Roche, Basel, Switzerland) was used to prepare whole-cell lysates. Briefly, equal amounts of lysate were separated by SDS-polyacrylamide gel electrophoresis and then transferred to PVDF membranes (Millipore,Massachusetts, USA). After blocking with 5% bovine serum albumin, the membranes were probed with anti-FGF9 or CCND2 and anti-GAPDH (ab71395, ab226972, ab9485, Abcam, Cambridge, UK), followed by incubation with a horseradish peroxidase–conjugated secondary antibody [goat-anti-mouse IgG (1:2000) and goat-anti-rabbit IgG (1:3000)]. The proteins were visualized using Image Reader LAS-4000 (Fujifilm) and analyzed with Multi Gauge V3.2 software (GE Healthcare Life Sciences, USA).

Protein samples were boiled in SDS/β-mercaptoethanol sample buffer, and 20 μg of each sample was loaded into each lane of 4–12% polyacrylamide gels. After separation by electrophoresis, the proteins in the gels were transferred onto PVDF membranes (Amersham Pharmacia Biotech, St. Albans, Herts, UK). The membrane was incubated with rabbit anti-CDK6 polyclonal antibody (ab151247, Abcam, Cambridge, MA, USA) or rabbit anti-EZH2 monoclonal antibody (ab191080, Abcam, Cambridge, MA, USA) or rabbit anti-PDL1 polyclonal antibody (ab205921, Abcam, Cambridge, MA, USA) or rabbit anti-TAK1 polyclonal antibody (ab109526, Abcam, Cambridge, MA, USA) or rabbit anti-KRAS polyclonal antibody (ab180772, Abcam, Cambridge, MA, USA), or rabbit anti-FGF9 polyclonal antibody (ab71395, Abcam, Cambridge, MA, USA) or mouse anti-β-actin monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) over night at 4 °C. The specific protein-antibody complex was detected by using horseradish peroxidase conjugated goat anti-rabbit or rabbit anti-mouse IgG. Detection by the chemiluminescence reaction was carried using the ECL kit (Pierce, Appleton, WI, USA). The β-actin signal was used as a loading control.

Primary HSCs were seeded on chambered coverglasses (Nunc LAB-TEK chamber slide system, ThermoFischer Scientific, Waltham, MA, USA) and stimulated as indicated. Cells were fixed with 4% parafolmaldehyde and treated with 0.2% Triton X-100. After blocking with 5% goat serum, cells were stained anti-FGF9 antibody (ab71395, Abcam).