Western Blot Analysis of Mammosphere Proteins

We followed the method of [34 (link)]. Proteins isolated from mammospheres treated with DHTS were separated on 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). PVDF membranes were blocked in 3% bovine serum albumin (BSA) in PBS-Tween 20 (0.1%, v/v) at room temperature for 1 hour. The PVDF membranes were incubated in blocking solution with primary antibodies at 4°C overnight. The primary antibodies used were Stat3, p65, lamin B (Santa Cruz Biotechnology), pStat3 (Cell Signaling, Beverly, MA, USA), and NOX2 (AB Frontier, Seoul, Korea). An antibody against β-actin (Santa Cruz Biotechnology) was used as a control. After PVDF membranes were washed with PBS-Tween 20 (0.1%, v/v), the membranes were incubated with HRP-conjugated secondary antibodies and enhanced using the chemiluminescence detection kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA).