Albumin bovine 5 bsa

MC3T3-E1 cells were sown in 48-well plates at a density of 6 × 10³ cells/well for three days under various circumstances to determine the expression and distribution of NRF2. Cells were treated with primary anti-NRF2 antibodies (CST12721T, Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C after being fixed with 4% paraformaldehyde and blocked with 1% Albumin Bovine V (BSA, Solarbio, China) for an hour. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Solarbio, China) for 10 min after the cells were treated with an Alexa Fluor^® 594-conjugated goat anti-rabbit IgG secondary antibody (SA00013-4, Proteintech, China) for 2 h in the dark. Fluorescence microscopy (Leica, Germany) was used to take the pictures. ImageJ software was used to evaluate the mean fluorescence intensity of NRF2.

The tissue sections were deparaffinized and 3% H₂O₂ was used to block the endogenous peroxidase. The sections were incubated in tris-buffered saline (TBS) with 5% albumin bovine V (BSA; Solarbio, Beijing, China) for 1 h. The cell samples were washed with PBS three times and fixed with 4% paraformaldehyde for 15 min. After washing with PBS three times, tissue sections or cells were permeated with 0.3% Triton X-100 for 15 min and then incubated with p21 antibody (1:200; Abcam, Cambridge, MA, USA), p16 antibody (1:200; Cell Signaling Technology, Danvers, MA, USA), LC3 antibody (1:200; CST, Danvers, MA, USA), PCNA antibody (1:200; Proteintech, Wuhan, China) or Ki67 antibody (1:200; Abways, Shanghai, China) in 5% BSA overnight at 4 °C. After washing with TBS, the sections were incubated with a Rhodamine (TRITC)-conjugated goat anti-rabbit IgG (H+L) (1:300; Abcam, Cambridge, UK). The nuclei were counterstained with DAPI (Invitrogen, Waltham, MA, USA). The sections were washed with PBS three times, and coverslips were mounted in 90% glycerol in PBS. The fluorescence was detected by a fluorescence microscope (Nikon, Tokyo, Japan). Image J was used to analyze the fluorescence intensity.

The treated cells were collected, washed twice by PBS and lysed with RIPA lysis containing protease and phosphatase inhibitors (Thermo Fisher Scientific) for 30 min on ice. The supernatant was loaded and boiled in water for 10 min. 20 µg/lanes of protein were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and then transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany). The transblotted membranes were blocked for 2 h using 5% nonfat milk for non-phosphorylated protein or 5% Albumin Bovine V (BSA, Solarbio, China) for phosphorylated protein. The blocked membranes were incubated with corresponding primary antibodies at 4 °C overnight. Then, the membranes were washed three times for 15 min each time with TBST, followed by the appropriate second horseradish peroxidase (HRP)-conjugated antibody for 1 h at room temperature. After washing three times again for 15 min with TBST, the protein bands were detected by the Chemi Doc TM MP Imaging System (Bio-Rad). The primary antibodies, including anti-caspase-3, -BCL2, -BCL-X_L, -MCL1, -P21, -P27, -cyclin D1, -cyclin-E1, -p-ATM, -p-ATR-, -p-CHK1, -p-CHK2 and -GAPDH were obtained from Cell Signaling Technology (MA, USA). Anti-CDK4, -CDK6, -PARP1, -γH2A.X, -KU80 and -Rad51 antibodies were purchased from Abcam (Cambridge, UK).