Western Blot Analysis of Apoptosis and MAPK Signaling

Total proteins from BC cells were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer containing phenylmethyl sulfonylfluoride (PMSF), followed by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After that, the PVDF membranes were blocked with 5% skimmed milk and incubated with the corresponding primary antibody: Bax (1:1000, Abcam, Cambridge, UK), Bcl-2 (1:1000, Abcam), MAPK6 (1:1000, Abcam), p-MAPK6 (1:1000, Abcam), p38 (1:1000, Cell Signaling Technology, Danvers, MA, USA), p-p38 (1:1000, Cell Signaling Technology), ERK (1:1000, Cell Signaling Technology), p-ERK (1:1000, Cell Signaling Technology), and β-actin (1:5000, Cell Signaling Technology). Next, the membranes were incubated at 4℃ for 14 h, and subsequently, the membranes were incubated with the corresponding secondary antibody (1:6000, Cell Signaling Technology) for 1.5 h at room temperature. Lastly, the band signal was visualized using an enhanced chemiluminescence (ECL) detection system (Millipore).