Westfemto chemiluminescent reagent

Acinar cells were isolated and seeded into 6-well tissue culture plates. The cells were treated with 250 µM hydrogen peroxide for 7.5 min with or without 10 and 30 µM TCT pretreatment (1 h). Total protein lysates were separated on 8% SDS-PAGE gels at 100 V for 90 min. Proteins were transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in phosphate buffered saline with Tween^® 20 (PBST) for 1 h at room temperature. Membranes were incubated with antibodies against PAR (clone 10H; purified in-house) overnight at 4 °C. After washing with Tris buffered saline with Tween^® 20 (TBST), membranes were incubated with peroxidase-conjugated anti-mouse IgG (1:3000; Cell Signaling, Danvers, MA, USA) and anti-β-actin (1:20,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies for 2 h at room temperature. Membranes were then washed with PBST and incubated with WestFemto chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA, USA). Luminescence was detected with a Chemidoc Touch gel documentation system (BioRad Laboratories, Hercules, CA, USA). Signal intensities were quantified using Image Lab software (BioRad). The blot area used for signal quantification was selected to cover the PARylated PARP1 region (ca. 100–180 kDa) and actin signals were used for normalization.

Western blotting was performed as described previously (Hassanian et al., 2015[12 (link)]). Following treatment with Tranilast (200 µM), Cyclin D1 protein was extracted using lysis buffer. We evaluated protein concentration using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL). Following sample loading and separation by SDS-PAGE, proteins were transferred onto poly vinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA). We incubated PVDF membranes with primary and secondary antibodies, and visualized data using a West Femto Chemiluminescent reagent (Thermo Scientific, Rockford, IL).