Western Blot Analysis of SE1 Effects

HT1080 cells were incubated with SE1 (10, 20, 50, and 100 μM) for 1 h and then incubated with PMA (10 ng mL⁻¹) for 24 h. Cells were collected and lysed in RIPA buffer. Equivalent amounts of proteins (20-40 μg) were separated by SDS-PAGE, as described previously [24 (link)], and subsequently transferred to NC membranes. Membrane was blocked with 5% skim milk at room temperature for 2 h and then incubated with primary antibodies. After being washed with TBST, the membranes were incubated with secondary antibodies for 2 h at room temperature and visualized with an enhanced chemiluminescence (ECL) detection system (Syngene, Cambridge, UK).