Anti lc3 antibody

HSC-2 cells were washed in ice-cold PBS and then whole cell lysates were prepared using laemmli sample buffer. Whole cell lysates (5 ÿ 10⁴ cells per lane) were separated by 12% SDS-polyacrylamide gel electrophoresis, and blotted onto a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membrane was then blocked against non-specific binding by treatment for 1 h with 5% bovine serum albumin in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20, and then immunoblotted overnight at 4 °C using the respective primary antibody. Next, the membrane was incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody, and the protein bands were visualized using an immobilon Western Chemiluminescent HRP Substrate (Merck Millipore). Protein expression was quantified using ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). The following antibodies were used: anti-PARP, anti-caspase 3 antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-LC3 antibody (1:1000, Medical & Biological Laboratories, Nagoya, Japan), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:20,000, Trevigen, Helgerman, Gaithersburg, MD, USA), and anti-rabbit IgG horseradish peroxidase-linked whole antibody (1:20,000, GE Healthcare, Little Chalfont, Buckinghamshire, England).