Alphaimager hp system

Cell culture was grown overnight in TGY medium supplemented with antibiotics when necessary. The culture was then subcultured in fresh medium until OD₆₀₀ reached ~ 0.8. Two microliters of prepared culture was spotted in the center of TGY plates (supplementary with appropriate antibiotics) with 0.5% agar. The diameter of cell migration was measured after 12 h of cultivation. Pictures were taken with the AlphaImager^® HP system (Alpha Innotech, USA).

B16-F10 cells were treated with test samples in different concentrations to determine levels of melanogenic proteins, such as tyrosinase, TRP-1, TRP-2, and MITF. Besides, B16-F10 cells were treated with 50 μg/mL of test samples for indicated time to determine melanogenesis signaling pathways of p-ERK, p-JNK, and p-CREB. Cells were harvested with ice-cold modified radioimmune precipitation assay buffer, containing a protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA), 50 mM Tris, 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 10 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate. Cell lysates were centrifuged at 15,000 × g for 30 min at 4 °C, and the supernatant containing proteins was collected. Protein concentrations were determined by using the Bio-Rad protein assay kit. Equal amounts of protein were separated on the polyacrylamide gel and transferred to nitrocellulose blotting membranes. Membranes were blocked, incubated with the indicated primary antibody, washed, and incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized with the enhanced chemiluminescence reagent with the AlphaImager HP system (Alpha Innotech, USA). Bands in the immunoblots were quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

After 24 hours of treatment with A1 or vehicle, the M17 cells were harvested in 2 x SDS-PAGE sample buffer. The samples were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes. Then, the membranes were blocked in 5% milk for one hour, followed by incubation overnight at 4°C with the corresponding primary antibody. The primary antibody was anti-APP, which is specific to the APP amino terminal (Abcam, Cambridge, UK), and anti-BACE1 (Sigma Aldrich, St. Louis, MO, USA). On the subsequent day, the membranes were washed three times with TBST buffer for five minutes. The membranes were then incubated in 5% milk (v/v) supplemented with anti-rabbit IgG or anti-mouse IgG for one hour at room temperature. Following incubation with the secondary antibody, the membranes were washed three times with TBST buffer for five minutes. The blots were then visualized via incubation with a SuperSignal West Dura chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA). Images were obtained via an AlphaImager HP System (Alpha Innotech, San Leandro, CA, USA). β-actin was used as a reference control for protein loading. The bands were quantified using Fluorchem Q SA software (Alpha Innotech, Santa Clara, CA, USA).