Western Blot Analysis of SKOV3 Cells

Proteins were extracted from SKOV3 cells treated with rfhSP-D for 24 and 48 h (21 (link)) and separated on a 10% v/v SDS-PAGE. The separated proteins were then electrophoretically transferred onto a nitrocellulose membrane (Thermo Scientific) in Wet-Transfer Buffer. The membrane was incubated in TBS containing 5% w/v dried milk powder and 0.1% v/v Tween Tween-20, for 1 h at room temperature to block non-specific binding. The membrane was then incubated with primary anti-human SP-D polyclonal antibodies (raised in rabbit), caspases 3 and 9, and phospho (Thr389) p70S6K (Cell Signaling Technology) at 4°C overnight. The membranes were washed in TBS + 0.1% Tween-20 (3 times, 15 min each) before incubation with the secondary goat anti-rabbit IgG-HRP-conjugated antibody for 1 h at room temperature. Proteins were visualized as previously described (21 (link)).