Anti ago2 antibody

RAW264.7 cells were seeded in 6-well plates (2×10⁵ cells per well) and cultured overnight. The following day, cells were treated with SjEVs (5 μg/well) or NCTC clone 1469 cell EVs (5 μg/well). Cell lysates were prepared in 500 μL of lysis buffer containing 25 mM Tris (pH 7.4), 150 mM KCl, 0.5% NP-40, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 M NaF, 0.5 M dithiothreitol, and protease inhibitors and then centrifuged at 10,000×g for 10 min at 4 °C. The pull-down assay was performed using a Dynabeads Protein G Immunoprecipitation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions with Anti-AGO2 antibody (Abcam, Catalog number: ab186733). In brief, approximately 10 μL of Anti-AGO2 antibody was coupled with 1.5 mg of Dynabeads. Then, 200 μL of the protein lysate (100 μg of protein) was incubated with the antibody-treated beads for 20 min at room temperature, and the Dynabeads were washed three times in 200 μL of the wash buffer provided with the kit. Finally, target antigens were eluted with elution buffer, the elutes separated by SDS-PAGE, and then analyzed by immunoblotting using Anti-AGO2 antibody and tubulin antibody (Catalog number: T6074, Sigma). RNA was also isolated from the immunoprecipitates and qPCR was used to examine SjEV miR-125b and bantam levels.

RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation (RIP) Kit (EMD Millipore). U251 and T98G cells transiently transfected with miR-205-3p were harvested using RIP lysis buffer and 100 μl of the cell lysate was employed for RIP experiments using an anti-AGO2 antibody (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. The beads were attracted by a magnetic separator, and samples were fixed with proteinase K. The RNA fraction isolated by RIP was subjected to qRT-PCR analysis to identify the direct binding between linc00645 and miR-205-3p.

RIP was carried out with the Magna RIP RNA binding protein immunoprecipitation kit (Millipore, Bedford, MA, USA). BC cells transfected with miR-195-5p mimics or miR-NC at 80% confluence were collected, then lysed using RIP lysis buffer. Then, the anti-Ago2 antibody (Abcam, Shanghai, China) was applied for Ago2 immunoprecipitation, and the immunoglobulin G (IgG) antibody served as a negative control. Immunoprecipitated RNA was separated and the enrichment of CircWHSC1 and FASN in the lysates was assessed by qRT- PCR.