Protein lysate buffer

Kaempferol and kaempferide were isolated from Hippophae rhamnoides L., as previously described [20 (link),56 (link)]. OA, oil red O and sulforhodamine B (SRB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Gibco (Carlsbad, CA, USA). Fetal Bovine Serum (FBS) was from Zhejiang Tianhang Biological Technology Co., Ltd. Kits of measurement of triglyceride (TG) and superoxide dismutase (SOD) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). BCA assay kit and protein lysate buffer were obtained from Beyotime (Shanghai, China). Polyclonal antibodies against FAS, SCD-1, SREBP1, PPARγ, C/EBPα, Perilipin-1, Caveolin-1, Nrf2, HO-1 and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA).

Silibinin, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), JC-1, dimethyl sulfoxide (DMSO), and H₂O₂ (30%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). MitoSOX^TM Red was from ThermoFisher Scientific (Rockford, IL, USA). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). Kits for measurement of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Bicinchonininc acid (BCA) assay kit and protein lysate buffer were obtained from Beyotime (Shanghai, China). Polyclonal antibodies against nuclear factor 2-related factor 2 (Nrf2, #12721), heme oxygenase-1 (HO-1, #26416), extracellular regulated protein kinases (ERK, #4695, p-ERK, #4370), sirtuin 1 (SIRT1, #2493), dynamin-related protein 1 (Drp1, #8570), optic atrophy 1 (OPA1, #67589), and β-actin (#4970) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Cell, retina and RPE/choroid mixture samples were sonicated in protein lysate buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor cocktails (Beyotime, Shanghai, China). A bicinchoninic acid assay was used to measure the protein concentration. An equal amount (20 μg) of cell lysate was dissolved in the sample buffer, after which samples were boiled for 6 min. After denaturation, electrophoresis was performed with 10% polyacrylamide gels containing 0.1% SDS, and then proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h at room temperature and rinsed in TBS-T three times. Then the membranes were subsequently incubated with the specific primary antibody overnight at 4°C. The membranes were washed three times with TBS-T and incubated with the corresponding biotinylated secondary antibodies for 1 h at room temperature. Signals were subsequently developed using enhanced chemiluminescence, after which images were captured using a microscope equipped with a CCD camera (Tanon, Shanghai). Finally, the band density of proteins was calculated with the ImageJ software (v1.51, NIH, USA).