Cleaved caspase 8

A549 cell lysates were prepared by harvesting, washing in cold PBS, resuspending in lysis buffer followed by sonication. Proteins (35 μg) were resolved by 10%–15% SDS gels and transferred to a nitrocellulose membrane, and analyzed by western blotting as described previously [65 (link)]. The following antibodies were used for immunoblotting: LC3(Novus Biologicals, Littleton, CO, USA), DR-4, DR-5, and β-actin Sigma-Aldrich (St. Louis, MO, USA), p62 (Millipore Corp., Milford, MA, USA), ATG5, cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-8 (BD pharmingen, USA), PPARγ Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Cells were harvested, washed in cold PBS, resuspended in lysis buffer [25 mM HEPES (pH 7.4), 100 mM EDTA, 5 mM MgCl₂, 0.1 mM DTT, and a protease inhibitor mixture], and sonicated to prepare A549 cell lysates. Proteins (35 μg) were separated in 10–15% SDS gels and transferred to nitrocellulose membranes. After incubation with 1:1000 primary antibody in dilution buffer (1% milk with PBS-Tween) and secondary antibody, Membranes were developed with enhanced chemiluminescence. The following antibodies were used for immunoblotting: LC3 (Novus Biologicals, Littleton, CO, USA), anti-P62 (Millipore Corp., Milford, MA, USA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-8 (BD pharmingen, USA), c-FLIP (Enzo life sciences, USA), DR-4, DR-5, and β-actin Sigma-Aldrich (St. Louis, MO, USA). Images were obtained using a Fusion-FX7 imaging system (Vilber Lourmat, Marne-la-Vallée, France).

A549 cell lysates were prepared by harvesting, washing in cold PBS, resuspending in lysis buffer followed by sonication. Proteins (35 μg) were resolved by 10%–15% SDS gels and transferred to a nitrocellulose membrane, and analyzed by western blotting as described previously [26 (link)]. The antibodies were used : LC3 (Novus Biologicals, Littleton, CO, USA), DR-4, DR-5, and ß-actin Sigma-Aldrich (St. Louis, MO, USA), p62ATG5, cleaved caspase-3(Cell Signaling Technology, Danvers, MA, USA), c-FLIP (Enzo life sciences, USA), cleaved caspase-8 (BD pharmingen, USA).