Western blot was carried out as described previously.22 (link)
Briefly, after treatment with TGF-β1 in the absence or presence of hesperetin for 48 hour, MDA-MB-231 cells were collected in a RIPA buffer (Wako, Japan) added with protein inhibitors by using a cell scraper. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked using 5% non-fat milk solution, then were treated with primary antibodies at 4°C overnight. Next, the membranes were washed 3 times in TBS-T and incubated with secondary antibodies for 1 hour at room temperature. Finally, Super Signal West Pico chemiluminescence substrates (Thermo Fisher Scientific, Waltham USA) were used to visualize the stained proteins. The protein bands were evaluated using Quantity One with ChemuDoc XRS-J software (Bio-Rad, USA).
Briefly, after treatment with TGF-β1 in the absence or presence of hesperetin for 48 hour, MDA-MB-231 cells were collected in a RIPA buffer (Wako, Japan) added with protein inhibitors by using a cell scraper. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked using 5% non-fat milk solution, then were treated with primary antibodies at 4°C overnight. Next, the membranes were washed 3 times in TBS-T and incubated with secondary antibodies for 1 hour at room temperature. Finally, Super Signal West Pico chemiluminescence substrates (Thermo Fisher Scientific, Waltham USA) were used to visualize the stained proteins. The protein bands were evaluated using Quantity One with ChemuDoc XRS-J software (Bio-Rad, USA).
