Goat anti rabbit secondary antibody

Western blot analysis was conducted as previously reported^{37 (link)}. Briefly, total protein was extracted from cell culture samples using RIPA buffer (1X RIPA, Catalog #9806, Cell Signaling Technologies, Danvers, MA). Protein concentration in each sample was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA). The same amount of protein (30 µg) was subjected to 12% Bis (2-hydroxyethyl) amino-tris (hydroxymethyl) methane (w/v) gel electrophoresis and electroblotted onto nitrocellulose membranes (GE Healthcare, Pittsburgh, PA). Rabbit anti-DROSHA (ab183732; 1:10,000; Abcam Inc., Cambridge, MA) and goat anti-rabbit secondary antibody (1:5000; GE Healthcare) were used. Stripping and re-probing for beta-actin (ab8227; Abcam; 1:10,000 dilution) was conducted to normalize DROSHA protein expression levels. Immunodetection was carried out using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, Waltham, MA).

Twenty microgram of protein was prepared with protein loading dye containing reducing agent and boiled for 3 min. Protein samples were separated by SDS-PAGE on 10% Tris-HCl protein separation gels, and transferred to Immobilon PSQ 0.2 μm PVDF membranes (Millipore). Membranes were blocked in TBST containing 5% milk, and incubated overnight at 4°C with primary antibody diluted in TBST containing 1% milk. Primary antibodies used: anti-GAPDH (1:5000, CST, D16H11), anti-PARP1 (1:1000, CST, 46D11), anti-Axin2 (1:400, Avivasysbio, OAAB02831). The following day, membranes were washed, incubated for 1 h at 20°C with goat anti-rabbit secondary antibody (1:2000, GE) diluted in TBST containing 1% milk, washed, and imaged on a ChemiDoc^TM Touch system (Bio-Rad).