Ripa buffer

Total cellular proteins were collected through the lysis of harvested cells with RIPA buffer (GeneTex Inc., Hsinchu City, Taiwan). Afterward, 25 μg of proteins was separated via SDS-PAGE and transferred onto PVDF membranes (Immobilon-P, Merckmillipore, Danvers, MA, USA). The membranes were blocked with 5% skimmed milk, incubated with primary antibodies at 4 ºC overnight, and incubated with horseradish peroxidase (HRP)-conjugated specific secondary antibodies at room temperature for 1 h. The signals were developed by incubating with an enhanced chemiluminescence substrate (PerkinElmer, Waltham, MA, USA) and captured with a luminescent image analyzer (Fusion Solo, Vilber Lourmat Deutschland GmbH, Germany). The following antibodies were used in this study: mouse monoclonal IgG anti-Myc tag antibody (Proteintech Group Inc., Rosemont, IL, USA); mouse monoclonal IgG anti-NQO1 (Cat. No. sc-32793), anti-Nrf2 (Cat. No. sc-365949), and anti-JNK (Cat. No. sc-7345) antibodies (Santa Cruz Biotechnologies Inc., Dallas, TX, USA); anti-phosphorylated JNK antibody (Cat. No. 4668S; Cell Signaling Technology, Danvers, MA, USA); rabbit polyclonal IgG anti-GAPDH antibody (Cat. No. GTX100118; GeneTex Inc., Hsinchu City, Taiwan); and mouse monoclonal IgG anti-β-actin (Cat. No. A5441; Sigma-Aldrich, St. Louis, MO, USA).

Total cellular proteins were obtained by lyzed with RIPA buffer (Genetex Inc., Irvine, CA, USA) and the protein concentration was quantified with the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). An amount of 25 µg of total cellular proteins was separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane (Pall Corporation, Washington, NY, USA). After blocking with 1% skim milk/TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) at room temperature (RT) for 1 h, the membrane was incubated with primary antibody at 4 °C overnight followed by horseradish peroxidase conjugated secondary antibody at RT for 1 h. The signal was then developed by Pierce™ ECL Western Blotting Substrate (Thermo Fisher) and captured by the FUSION Solo S imaging system (Marne-la-Vallée, France). The antibodies used in this study were listed in Table S1.