Porin

For immunoblot analysis 50μg of isolated hepatocytes lysate was resolved by a 4–20% gradient gel, transferred to a nitrocellulose membrane, and blotted with the appropriate primary antibodies. Membranes were incubated with peroxidase-conjugated secondary antibody (Cell signaling, Danvers, MA, USA). Protein bands were visualized using an enhanced chemiluminescence reagent and digitized using a CCD camera (ChemiDoc®, BioRad, Hercules, CA, USA). A rabbit anti-cleaved caspase 3, anti-caspase 3, anti-phospho-MYPT1, or anti-MYPT1 was purchased from Cell Signaling and anti-beta actin was purchased from GeneTex (Irvine, CA, USA). Protein load was verified using GAPDH (GeneTex), or PORIN (GeneTex) antibody.

For immunoblot analysis 50μg of whole-liver lysate, as well as mitochondria or cytosolic fraction with mitochondria isolation kit (Thermo scientific, Rockford, IL, USA), was resolved by a 4–20% gradient gel, transferred to a nitrocellulose membrane, and blotted with the appropriate primary antibodies. Membranes were incubated with peroxidase-conjugated secondary antibody (Cell signaling, Danvers, MA, USA). Protein bands were visualized using an enhanced chemiluminescence reagent and digitized using a CCD camera (ChemiDoc®, BioRad, Hercules, CA, USA). Expression intensity was quantified by ImageLab (BioRad). A rabbit anti-Bid, anti-BAX, anti-BAK, anti-cytochrome C, anti-cleaved caspase 3, anti-caspase 3, anti-cleaved caspase 8, or anti-caspase 8 antibody was purchased from Cell Signaling and anti-α-SMA and anti-GAPDH were purchased from GeneTex (Irvine, CA, USA). Protein load was verified using GAPDH (GeneTex), or PORIN (GeneTex) antibody.