Western Blot Analysis of ZFP-1 in Adult Worms

Adult worm lysates were resolved via SDS-PAGE on a 4 to 12% gel (Invitrogen NuPAGE Bis-Tris gel) and transferred to a 4.5-μm nitrocellulose membrane (Bio-Rad) at 100 mA for 1 h. The membrane was blocked using 3% (wt/vol) BSA in PBS with 0.01% Tween for 1 h, followed by overnight incubation at 4°C with an anti-ZFP-1 C-terminus-specific antibody [82 (link)], diluted 1:2,000 in PBST-3% BSA. The membrane was then washed 3 times with PBST, incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (PerkinElmer) diluted 1:5,000 in PBST-3% BSA, and visualized by using SuperSignal West Pico chemiluminescence substrate (Thermo Scientific) and a series 2000A film processor (Tiba).