Begin by adding two milliliters of MCF-7 cells containing different drugs into the wells of a six-well plate. Incubate the cell cultures for 24 hours at 37 degrees Celsius and 5%carbon dioxide. Harvest the cells by centrifugation at 560 x g for three minutes at four degrees Celsius.
Then wash the cells twice with pre-cool PBS, centrifuging at 560 x g for three minutes between the washes. Add 50 microliters of lysis buffer to the washed cells and place the sample in an ice bath for 15 minutes. Then centrifuge the sample at 8, 550 x g for 10 minutes at four degrees Celsius, and collect the supernatant protein sample.
Combine the protein sample with the loading buffer in a four to one ratio. Next, denature the mixture at 100 degrees Celsius for 10 minutes in a metal bath. Then cool the mixture at room temperature.
Separate the protein sample using 10%SDS polyacrylamide gel electrophoresis. Transfer the proteins onto a 0.22 micrometer PVDF membrane. After blocking the membrane with 5%BSA, incubate it with the corresponding primary antibodies overnight at four degrees Celsius.
The following day, incubate the membrane with goat anti-rabbit IgG secondary antibody at 37 degrees Celsius for two hours. Then develop the membranes using an ECL chemiluminescence solution and capture the images using a contactless quantitative Western blot imaging system. Western blot showed that salidroside treatment promoted the protein expression of the pro-apoptotic factors CC-9, CC-7, CC-3, Bim, and Bax while it inhibited the protein expression of anti-apoptotic BCL-2.
Salidroside prominently limited the ratios of p-PI3K to PI3K and p-AKT to AKT. Meanwhile, the protein expression of mTOR, HIF-1 alpha, and Fox01 was also notably suppressed.
