Anti pycr1

The infected cells were collected, washed twice with PBS, and lysed in lysis buffer. After lysed on ice for 15 min, the cells were broken by sonication. Then, the samples were centrifuged at 10000 g for 15 min at 4 °C to collect the supernatant. The protein concentration was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime Biotech Co., Ltd, Shanghai, China). The proteins were separated by 10% SDS-polyacrylamide gel and electro-transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature and incubated with primary antibody overnight at 4 °C. And then the samples were incubated with the respective second antibody (1:2,000) for 1.5 hours. The protein signal was visualized using the enhanced chemiluminescence kit (ECL-plus; Thermo). GAPDH was used as an internal control.
The antibodies used in this study were as follows: Anti-PYCR1 (No. ab103314; 1:300; Abcam, CA, USA), Anti-GAPDH (No. sc-32233; 1;2,000; Santa Cruz, TX, USA), Anti-Mouse IgG (No. #7076; 1:2,000; Cell Signaling Technology, CA, USA), Anti-Rabbit IgG (No. #7074; 1:2,000; Cell Signaling Technology, CA, USA).

(+)-Apl-1 was provided by Instituto Biomar (León, Spain) and was dissolved in DMSO and stored at −20 °C. Supplements and other chemicals not listed in this section were obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Cell culture media, penicillin, streptomycin and amphotericin B were purchased from Biowhittaker (Walkersville, MD, USA). Fetal bovine serum (FBS) and human serum were products of Harlan-Seralb (Belton, UK). Antibodies (anti-TXNRD1, anti-TXNDC5, anti-PYCR1, and anti-PRX IV) were acquired from AbCam (Cambridge, UK). Antibodies (anti-GAPDH, anti-p-Iκκα/β, anti-Iκκβ) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies (anti-Nrf2) were purchased from Santa Cruz Biotechnologies (Dallas, TX, USA). Secondary antibodies (anti-rabbit IgG, horseradish peroxidase-linked whole antibody, and anti-mouse IgG, horseradish peroxidase-linked whole antibody) were acquired from GE Healthcare (Buckinghamshire, UK). Plastics for cell culture were supplied by NUNC (Roskilde, Denmark) and VWR (West Chester, PA, USA).