Anti ago2 clone 4g8

Whole cell extracts or immunoprecipitated protein complexes were resolved by electrophoresis using SDS-10% polyacrylamide gel (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes (Thermo Fisher Scientific, Waltham, MA). Membranes were blocked with 5% milk in Tris-buffered saline/Tween-20 for 1 hr at room temperature, and then probed overnight at 4°C with pan-specific anti-NFI (Cat #sc-74444), anti-Ago1 (Cat #sc-376696), anti-HuR (Cat #sc-5261),anti-CUGBP1 (Cat #sc-56649) (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-Ago2 (clone #4G8; Wako, Richmod, VA) antibody. After washing, blots were incubated with the appropriate HRP-conjugated secondary antibody for 2 hr at room temperature. Proteins were detected with the enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA), the bands were visualized using the ChemiDoc XRS System (Bio-Rad), and the images were captured with the Image Lab Software V3.0. Membranes were stripped and reprobed with β-actin antibody (Santa Cruz) as a loading control.

For CLIP, HUVECs were irradiated with UV light at 400 mJ/cm² to crosslink RNA and proteins. Cells were then lysed in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM EDTA, and 100 U/μl RNase inhibitor. The lysates were incubated with protein G Dynabeads conjugated with anti-AGO2 (clone 4G8; Wako Chemicals) at 4°C overnight. Mouse IgG was used as an isotype control. The immunoprecipitated RNAs were then extracted with Trizol. For ChIP, HUVECs were treated with 0.75% formaldehyde for 20 minutes at room temperature. After sonication, the SREBP2-bound chromatin was immunoprecipitated by rabbit anti-SREBP2(N) (Abcam) conjugated to protein A Dynabeads. Protein and RNA were degraded by proteinase K and RNase A, respectively. The purified chromatin DNA was then used as the template for qPCR. As an isotype control, rabbit IgG was used in ChIP.