Ecl plus immunoblotting detection system

For Western blot analysis, 5 μg of total protein from soluble extracts were separated by 12% acrylamide SDS-PAGE gels, transferred to nitrocellulose membranes (Bio-Rad), and probed with AnNTRC (1:1000), 2-Cys Prx (1:5000) and TrxA (thioredoxin A) (1:3000) antibodies. In order to detect High Molecular Mass (HMM) species 10% acrylamide non-reducing SDS-PAGE (where reducing agents were avoided in the loading buffer) and native-PAGE were used. Signals were detected using an anti-rabbit secondary antibody (Sigma-Aldrich, St Louis, MO, US) and the ECL-Plus immunoblotting detection system (GE Healthcare, Buckinghamshire, England).

A2058 and MEG-01 cells were plated and grown overnight on 6-well plates. For sample preparation, cells were lysed with RIPA lysis buffer (EMD Millipopre Corp., Billerica, MA, USA) supplemented with a protease inhibitor cocktail. Whole-cell lysates were centrifuged at 13,000× g for 20 min at 4 °C and the concentrations of supernatant protein samples were determined with a Bradford protein kit assay (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (70 μg protein/lane) were loaded and separated by 4–12% Tris-glycine precast gel (Koma Biotech, Seoul, Korea). The gel was then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were washed with TBST (Tris-buffered saline with 0.1% tween 20) and incubated in blocking buffer (5% skim milk in TBST) at room temperature for 1 h. The membranes were incubated overnight at 4 °C with primary antibodies specific for PAR1 and β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:1000). After three washes with TBST, the membranes were incubated for 1h at room temperature with an anti-mouse secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:5000). The membranes were washed 3 times with TBST for 5 min each and then developed using the ECL Plus immunoblotting detection system (GE Healthcare, Piscataway, NJ, USA).

For Western blot analyses, cells were plated on 6-well plates and were serum-starved overnight. Cells were treated with compounds accordingly, and protein samples were prepared as described previously [36 (link)]. Then, 30 µg of total proteins was loaded to each well, and proteins were separated using 4–12% Tris Glycine Precast Gel (KOMA BIOTECH, Seoul, Republic of Korea). Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Blocking was carried out using Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% BSA at room temperature for 1 h. Then, the membranes were incubated with primary antibodies overnight at 4 °C with the indicated antibodies: anti-p42/44 (Cell Signaling; Cat#9102S) and anti-phospho-p42/44 (Cell Signaling; Cat#9101L). Subsequently, the membranes were washed out with TBST 3 times at 5 min intervals and incubated with HRP-conjugated anti-secondaries for 1 h at room temperature. Finally, visualization was carried out using an ECL Plus immunoblotting detection system (GE Healthcare, Piscataway, NJ, USA). All experiments were repeated five times independently, and ImageJ software (NIH, Bethesda, MD, USA) was used for analysis.