Beyoecl star system

PU (CAS: 65995-63-3, purity = 78% for animal treatment and 98% for cell treatment) was acquired from Chengdu Herbpurify Co. Ltd. (Chengdu, China). CCl₄ was purchased from Tianjin Guangfu Technology Development Co. Ltd. (Shanghai, China). Vegetable oil purchased from Hunan Jinhao Camellia Oil Co. Ltd. (Hunan, China). Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and lactate dehydrogenase (LDH) detection kits were provided by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Proteins were extracted using a tissue protein extraction kit (Beyotime Biotechnology, Beijing, China). MTT assay kits were purchased from Ding Guo Changsheng Biotechnology Co. Ltd. (Beijing, China).
Antibodies against P62 (#18420-1-AP), Beclin1 (#11306-1-AP), LC3B (#18725-1-AP), Nrf2 (#16396-1-AP), Akt phosphorylation (p-Akt) (#66444-1-Ig), and β-actin (mouse polyclonal) were purchased from Proteintech, Inc. (Wuhan, China). Antibodies against phosphorylation of FOXO3a (p-FOXO3a) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Secondary antibodies were purchased from ZSGB-BIO Biotechnology Co. Ltd. (Beijing, China). Membrane blots were developed using the BeyoECL Star system (Beyotime Biotechnology).

Protein samples were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blocked with 5% nonfat milk solution. The membranes were cut into strips according to molecular weight of target protein, and each strip was incubated separately with primary antibody for target protein overnight followed by corresponding secondary antibody for 1 h. Primary antibodies to HMGB1, Lamin-B1, Tubulin, and CACNA1H were purchased from Abcam while other antibodies were obtained from Cell Signaling Technology. Antibody binding was visualized using the BeyoECL Star system (Beyotime, China).

The whole cell lysates were prepared by using RIPA lysis buffer containing PMSF (Beyotime, China). The cytosolic and nuclear fractions were prepared as aforementioned. Protein samples were separated using 12% SDS-PAGE gel, transferred to polyvinylidene difluoride membranes, and blocked with 5% nonfat milk solution. The membranes were incubated with primary antibody overnight followed by the corresponding secondary antibody for 1 h. Antibodies were obtained from Abcam (Shanghai, China) and listed as follows: rabbit anti-HMGB1 IgG, rabbit anti-Lamin B1 IgG, rabbit anti-CXCR4 IgG, rabbit anti-tubulin IgG, and horseradish peroxidase-conjugated goat anti-rabbit IgG. Tubulin served as the internal control for whole cell lysates and cytosolic fractions. Lamin B1 served as the internal control for nuclear fractions. Antibody binding was visualized using the BeyoECL Star system (Beyotime, China).