Bax polyclonal antibody

Western blot analysis of total protein isolated from NRCMs was performed as previously described 13. Briefly, protein samples (30 or 50 μg) were separated in 10% and 15% SDS‐PAGE and blotted to PVDF membrane. The membranes were incubated with the primary antibodies including rabbit anti‐SCAD monoclonal antibody (diluted 1:1000; Abcam, Cambridge, UK), anti‐Caspase‐3 polyclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), Bcl‐2 polyclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), Bax polyclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), AMPK monoclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA), p‐AMPK monoclonal antibody (diluted 1:1000; Cell Signaling Technology, Danvers, MA, USA) and mouse anti‐PPARα monoclonal antibody (diluted 1:500; Sigma‐Aldrich, St.Louis, MO, USA), anti‐α‐tubulin monoclonal antibody (diluted 1:5000; Sigma‐Aldrich, St.Louis, MO, USA), and then the HRP conjugated goat‐antimouse or rabbit IgG1 secondary antibody (1:10,000; Sigma‐Aldrich, St.Louis, MO, USA) were used. Finally, the protein expression levels were detected with chemiluminescent substrate (Thermo, Waltham, MA, USA). Results were analysed with the Image J system.

HepG2, Hep3B, Huh7 and SNU449 were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum. Chang and Huh1 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum. To detect cytochrome c release, we prepared cytosolic and mitochondrial lysates of the cells. Briefly, the HCC cells were homogenized, and mitochondrial fractions were acquired by a mitochondria isolation kit (Thermo Fisher Scientific, Rockford, IL, USA). The primary antibodies used include: SAG polyclonal antibody (Abcam, Cambridge, MA, USA), Bcl-2 monoclonal antibody (Santa Cruz, Dallas, TX, USA), Bax polyclonal antibody (Cell Signaling, Danvers, MA, USA), Noxa polyclonal antibody (Santa Cruz), SARM monoclonal antibody (Cell Signaling), ubiquitin monoclonal antibody (Santa Cruz), GAPDH monoclonal antibody (Santa Cruz), VDAC polyclonal antibody (Cell Signaling), cytochrome c monoclonal antibody (BD Biosciences, San Jose, CA, USA), Alexa 488-conjugated secondary antibody (Invitrogen, Grand Island, NY, USA) and a loading control, β-actin (Sigma, St Louis, MO, USA).