AGS and HT-29 cells were seeded in a 6-well microtiter plate at
5×106 cells/mL in DMEM+10% FBS+1%
penicillin and incubated at 37°C in 5% CO2 incubator for
24 h. After the incubation, the cell medium was removed, and the cells were
washed with DPBS. The cells were treated with 10-fold diluted EE and then
incubated at 37°C for 3 h under 5% CO2. The medium was
removed, and the cells were washed with chilled (4°C) DPBS. The 200
μL of RIPA buffer (iNtRON Biotechnology, Seongnam, Korea) was added
to each well and placed on ice for 30 min to extract the protein from AGS
and HT-29 cells. The cell suspension was centrifuged at 15,814×g and
4°C for 15 min, and the supernatant was transferred to a
microcentrifuge tube. A DCTM Protein Assay (Bio-Rad Laboratories,
Hercules, CA, USA) was used to determine the concentration of the extracted
protein. Western blotting was used to measure protein expression as follows;
on a 12% SDS-PAGE, 30 μg of protein was separated at 120 V for 1 h.
Proteins were then transferred to a polyvinylidene difluoride (PVDF)
membrane (GE Healthcare Life Sciences, Marlborough, MA, USA) at 60 V for 2.5
h. After that, the membranes were treated with 5% skim milk (Sigma-Aldrich)
at 20°C for 1 h. In order to perform immunoblotting, primary
antibodies [{mouse-anti-BAX (ABM40273, 1:300) from Abbkine
Scientific, Hubei, China}, {mouse-anti-p53 (sc-100, 1:200)
from Santa Cruz Biotechnology, Dallas, TX, USA}, and
{rabbit-anti-p21 (ABP57266, 1:300) from Abcam, Cambridge, UK}]
were used. As a reference protein, mouse-anti-β-actin (sc-81178,
1:1,000, Santa Cruz Biotechnology) was used. The secondary antibody was goat
anti-rabbit IgG-HRP (SA002-500, 1:5,000, GenDEPOT). The immunoreactive bands
were visualised by an LAS-3000 Imager (Fujifilm, Tokyo, Japan), and GelQuant
software v.2.7. (DNR Bio Imaging Systems, Jerusalem, Israel) was used to
quantify the intensity of the bands.
5×106 cells/mL in DMEM+10% FBS+1%
penicillin and incubated at 37°C in 5% CO2 incubator for
24 h. After the incubation, the cell medium was removed, and the cells were
washed with DPBS. The cells were treated with 10-fold diluted EE and then
incubated at 37°C for 3 h under 5% CO2. The medium was
removed, and the cells were washed with chilled (4°C) DPBS. The 200
μL of RIPA buffer (iNtRON Biotechnology, Seongnam, Korea) was added
to each well and placed on ice for 30 min to extract the protein from AGS
and HT-29 cells. The cell suspension was centrifuged at 15,814×g and
4°C for 15 min, and the supernatant was transferred to a
microcentrifuge tube. A DCTM Protein Assay (Bio-Rad Laboratories,
Hercules, CA, USA) was used to determine the concentration of the extracted
protein. Western blotting was used to measure protein expression as follows;
on a 12% SDS-PAGE, 30 μg of protein was separated at 120 V for 1 h.
Proteins were then transferred to a polyvinylidene difluoride (PVDF)
membrane (GE Healthcare Life Sciences, Marlborough, MA, USA) at 60 V for 2.5
h. After that, the membranes were treated with 5% skim milk (Sigma-Aldrich)
at 20°C for 1 h. In order to perform immunoblotting, primary
antibodies [{mouse-anti-BAX (ABM40273, 1:300) from Abbkine
Scientific, Hubei, China}, {mouse-anti-p53 (sc-100, 1:200)
from Santa Cruz Biotechnology, Dallas, TX, USA}, and
{rabbit-anti-p21 (ABP57266, 1:300) from Abcam, Cambridge, UK}]
were used. As a reference protein, mouse-anti-β-actin (sc-81178,
1:1,000, Santa Cruz Biotechnology) was used. The secondary antibody was goat
anti-rabbit IgG-HRP (SA002-500, 1:5,000, GenDEPOT). The immunoreactive bands
were visualised by an LAS-3000 Imager (Fujifilm, Tokyo, Japan), and GelQuant
software v.2.7. (DNR Bio Imaging Systems, Jerusalem, Israel) was used to
quantify the intensity of the bands.
