Sa0001 2

HaCaT cells were harvested with protein lysis buffer. And the protein concentrations were assessed using bicinchoninic acid (BCA) Protein Assay kit (Thermo Fisher Scientific, Waltham, MA). Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 100 V for 2 h, and then transferred to nitrocellulose membranes. After blocking with 5% dried nonfat milk for 1 h at room temperature, the membranes were incubated with primary antibodies, such as anti-Nrf2 (1:1000, 16396-1-AP, Proteintech, China), anti-Bcl-2 (1:1000, A19693, Abclonal, China), anti-Bax (1:1000, A19684, Abclonal, China) or anti-β-actin (1:10 000, AC026, Abclonal, China), respectively, overnight at 4°C. Following three rinses, blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10 000, SA0001-2, Proteintech, China) for 1 h at room temperature. Then the chemiluminescence signal in the blots was detected with Hypersensitive Chemiluminescence Substrate kit (17046, Zenbio, China) using the ePhoto developer (GenScript, China). The relative expression of the target protein was assessed using the internal reference β-actin as the standard. The intensity was quantified by ImageJ software (National Institutes of Health, Germany).

Protein samples were prepared from MIN6 cells in RIPA lysis buffer containing protease and phosphatase inhibitors (Sigma-Aldrich, PPC1010). Proteins were resolved on Tris/glycine gel and transferred to PVDF membrane (Bio-Rad, Hercules, CA, U.S.A.; 1620177). The following primary antibodies were incubated overnight at 4°C: anti-MPV17 (Proteintech, 10310-1-ap), anti-cleaved caspase-3 (Cell Signaling Technology, 9664s), and anti-GAPDH (Proteintech, 10494-1-ap). Anti-rabbit and anti-mouse secondary antibodies conjugated horseradish peroxidase (Proteintech, SA0001-1, SA0001-2) were used. Specific proteins on the blots were detected using ECL plus substrate (Millipore, WBULS0500).

Samples were separated on 10% SDS-PAGE and subsequently transferred to the Immobilon-NC Transfer Membrane (Millipore, USA). The membranes were blocked with 5% milk in Tris-buffered saline overnight and subsequently incubated with the following primary antibodies for 2 h at room temperature: anti-p65(8243S, CST); anti-phospho-NF-κB p65 (3033S, CST), anti-IκBα (4812S, CST), anti-p-IκBα (2859S, CST), anti-β-actin (3700S, CST), anti-His (T0009, Affinity), anti-ADA polyclonal. After the incubation, membranes were washed three times and incubated with a secondary HRP conjugated goat anti-rabbit (SA0001-2, proteintech) or goat anti-mouse (RS0001, Immunoway) (at 1:2000 dilution) for 1 h at 37 °C. The signals corresponding to the proteins were detected by enhanced chemiluminescence (ECL) (Millipore, USA). All the blots were cut prior to hybridisation with antibodies.