Anti rabbit igg horseradish peroxidase linked whole antibody

and 10 mM DTT. After denaturing by heating to 95°C for 10 min, samples were resolved in a NuPAGE 4–12% Bis-Tris Gel (ThermoFisher Scientific; NP0321BOX). The protein was transferred to an Immobilon-FL PVDF Membrane (Millipore; IPFL00010) using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad; 1703940). After blocking with 5% non-fat dry milk (Lab Scientific, M0841), membrane was incubated with anti-HaloTag antibody (1:1000; Promega Corporation; G9281) and then with anti-Rabbit IgG Horseradish Peroxidase linked whole antibody (1:5000; GE Healthcare; NA943V). Membrane was visualized using Radiance Plus chemiluminescent HRP substrate (Azure Biosystems, AC2103) on Azure 300 imager (Azure Biosystems). After visualization, the membrane was stripped for 40 min with shaking using NewBlot PVDF Stripping Buffer (LI-COR; 928–40032). After blocking, membrane was incubated with anti-GAPDH (FL-335) rabbit polyclonal antibody (1:4000; Santa Cruz; SC-25778) and then with anti-Rabbit IgG Horseradish Peroxidase linked whole antibody (1:5000; GE Healthcare; NA943V). Membrane was visualized as described above.

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).