Chemidoc touch gel documentation system

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 blots were carried out as previously described [40 (link),41 (link)]. Briefly, cells were washed once in PBS and collected by scraping into 100 μL of ice-cold lysis buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1 mM PMSF, and protease inhibitors). The extracts were further lysed with sonication and the supernatants were collected after centrifugation. Protein concentrations were determined with the Pierce BCA reagent (Thermo Fisher, Budapest, Hungary). Proteins (30 μg/lane) were separated in 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) for 90 min. Primary antibodies against iNOS (rabbit monoclonal; Sigma-Aldrich, Budapest, Hungary) were applied overnight at 4 °C. After three washes in TBS containing 0.05% Tween 20, the secondary antibody (peroxidase-conjugated goat anti-rabbit IgG, Sigma-Aldrich, Budapest, Hungary) was applied for 1 h. Blots were washed in TBS containing 0.05% Tween 20 three times and once in TBS. After washing, the blots were incubated in Pierce Supersignal Chemiluminescent reagent (Thermo Fisher, Budapest, Hungary). For detection of luminescence, Chemidoc Touch gel documentation system (BioRad) was used and signals were quantified by densitometry using the ImageLab 6.0 software.

Protein lysates extracted from L6, LHCN-M2 cells and human tissue were resolved using 8–12% SDS-PAGE and transferred to nitrocellulose or polyvinylidene difluoride membranes (PVDF) for immunoblotting. Immunoreactive bands were detected using enhanced chemiluminescence (ECL) or ECL prime (Amersham ECL Western Blotting detection reagent, GE Healthcare, Buckinghamshire, UK) reagents and imaged using a Chemi-Doc Touch gel documentation system (Bio-Rad, Hercules, CA, USA). Quantitation of signals was performed using Image Lab v. 5.1 software and normalized to Ponceau or GAPDH levels. Antibody details are provided in Supplementary Table S2.