Chemiluminescence substrate

The total protein separated by SDS-PAGE were transferred onto polyvinylidene difluoride (PVDF) membranes. Then the PVDF membrane was blocked with non-fat milk powder (1 h at room temperature), and the primary antibody (4°C for overnight) and secondary antibody (37°C for 1 h) were incubated. The primary and secondary antibodies were shown in Table 2. The gray value of each band was detected by chemiluminescence substrate (Boster, China).

Cell samples were washed with PBS, and the cell lysate was prepared using RIPA buffer containing a protease inhibitor cocktail (Roche Diagnostics GmbH, Germany) and a protein phosphatase inhibitor cocktail (Sigma, Merck KGaA, Darmstadt, Germany). Protein concentrations were identified by using a BCA kit (Sangon Biotech Co., Ltd., Shanghai, China). The cell lysate (50 μg) was resolved using 10% or 12% SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membrane. Following blocking in 1% skim milk, the PVDF membrane was incubated with the primary antibody at 4 °C overnight, according to the product datasheets. Subsequently, the membrane was washed with PBST (PBS buffer containing 0.1% Tween-20) or TBST (NaCl, Tris-HCL, 0.1% Tween-20) and incubated with the corresponding HRP-conjugated secondary antibody for 2 h at room temperature. Protein bands were visualized using a chemiluminescence substrate (Boster, Wuhan, China) and band density was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Polyvinylidene difluoride (PVDF) membranes were used to transfer the whole protein separated with SDS-PAGE. The primary antibody was added and incubated at 4 °C for overnight and the secondary antibody was incubated at 37 °C for an hour after non-fat milk powder blocked the PVDF membrane for an hour at RT. All antibodies we used were shown as below: HMGB1 (ab79823, Abcam, UK, 1:5000), Caspase-3 (ab184787, Abcam, UK, 1:2000), Bax (ab32503, Abcam, UK, 1:1000), Bcl-2 (ab182858, Abcam, UK, 1:2000), LRP (ab92544, Abcam, UK, 1:5000), MRP (ab260038, Abcam, UK, 1:1000), P-gp (ab170904, Abcam, UK, 1:1000), and the secondary antibodies (HRP Anti-Rabbit IgG antibody, Abcam, UK, 1:8000). Chemiluminescence substrate (Boster, China) was used to determine the gray value.