Western Blot Analysis of Proteins in MDA-MB-231 Cells

Total proteins in MDA-MB-231 cells were extracted by ice-cold RIPA cell lysis buffer (Beyotime, Shanghai, China). Total proteins (30 μg) of each sample were equally loaded onto SDS-PAGE electrophoresed gels and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% (w/v) skim milk at room temperature for 1 h, the membranes were incubated with primary antibody in Supplementary Methods, followed by incubation with secondary antibodies (1:2000) for 1 h at room temperature. Proteins on the PVDF membrane were visualized using chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). The intensities of the bands were corrected with respect to that of β-Actin. All the experiments were repeated three or more times^{34 (link),40 (link)}.