The protein levels of Nrf2, Beclin1, LC3, and p62 were quantified by semiquantitative densitometric analysis. Cell samples were lysed in RIPA, and a BCA protein assay kit was used to determine the protein concentration. Briefly, the total protein lysates were separated on 12% SDS-PAGE gels for detecting Nrf2, Beclin1, LC3, and p62. Then the proteins were transferred to polyvinylidene difluoride (PVDF) membranes, blocked with 5% skimmed milk for 2 h at room temperature, and then immunoblotted with rabbit polyclonal anti-human antibodies against β-actin (1 : 1000), Nrf2 (1 : 1000), Beclin1 (1 : 1000), LC3 (1 : 1000), and p62 (1 : 1500) overnight at 4°C. This was followed by application of a goat anti-rabbit peroxidase conjugated secondary antibody (1 : 5000, Santa Cruz) for 2 h at 37°C. Then the bands were detected using the enhanced chemiluminescence system and Quantity One image analysis software was used to measure the band intensity. The housekeeping protein β-actin was used for loading control. For result analysis, we normalized the band intensity of target protein to β-actin in each sample, and then we normalized the relative target protein expression level of treated group to its control group.
The cells were washed with phosphate-buffered saline (PBS) for three times, fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% Triton X-100 for 10 min, and then blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature. Next, cells were incubated with rabbit polyclonal anti-human antibodies against Nrf2 (1 : 150), Beclin1 (1 : 150), and LC3 (1 : 150) at 4°C overnight, respectively. After that, cells were washed and incubated with goat anti-rabbit dylight 594 (red) IgG antibody (QENSHARE BIOLOGICAL Inc., Xi'an, China) or goat anti-rabbit FITC (green) IgG antibody (ZSGB-BIO Inc., Beijing, China) at 1 : 150 dilution for 60 min at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole for 5 min. Cells were visualized with the fluorescent microscope (Nikon Eclipse Ti-s, Japan) using appropriate excitation wavelength. Full paper
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