Rabbit anti txnrd1

Cells were lysed with RIPA buffer and proteins were separated on 13% SDS-PAGE and transferred onto a PVDF membrane. The membrane was stained immunochemically using anti-KEAP1, NRF2, NQO1, TXNRD1 (Proteintech, Chicago, IL, USA), and by using anti-AKR1C1 (Abnova, Taiwan). H720 and H727 cells were cultured in RPMI 1640, supplemented with 10% FBS and antibiotics. Equal amounts of protein lysates (35 μg) were resolved by SDS-PAGE (13% acrylamide) under reducing conditions and transferred onto PVDF membranes (Immobilon PVDF, Millipore, Billerica, MA, USA). After transfer, PVDF membranes were blocked with 5% non-fat milk and incubated with primary antibody overnight at 4 °C. Rabbit anti-NRF2 (Proteintech), rabbit anti-NQO1 (Proteintech), rabbit anti-TXNRD1 (Proteintech), mouse anti-AKR1C1 (Abnova) and anti-Actin (Sigma-Aldrich, St. Louis, MO, USA) were incubated overnight at 4 °C. PVDF membranes were incubated with ECL plus solution following the manufacturer’s instructions (Thermo Fisher Scientific, Rockford, IL, USA). Chemiluminescence signals were acquired by ChemiDoc XRS (Biorad, Hercules, CA, USA).

Standard procedures were employed to do the Western blotting. Briefly, the cell lysates were prepared using RIPA lysis buffer supplemented with protease inhibitor cocktail, phosphatase inhibitor cocktail, and 1 mM PMSF (all from Sigma-Aldrich). Afterwards, samples were quantified using Bradford method. Same amount of protein was separated on 7.5–12% Bis–Tris gels (Epizyme, Shanghai, China) using MOPS/MES buffer. The electrophoretically isolated proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen, California, USA). Membranes were blocked for 90 min at 4 °C with 5% non-fat milk in PBST (0.1% Tween 20) and incubated overnight at 4 °C in solution containing relevant primary antibodies: mouse anti-GAPDH (Proteintech, Chicago, USA) 1:10000 and rabbit anti-TXNRD1 (Proteintech, Chicago, USA) 1:3000. Subsequently, horseradish peroxidase (HRP)-conjugated secondary antibody was diluted with PBST containing 3% non-fat milk and applied to the blots for an hour at room temperature. The ECL Chemiluminescence System (Tianneng, Shanghai, China) was implemented to examine antibody binding and analyzed using Image J software (version 2.0, LOCI, University of Wisconsin, Madison, WI, USA).