Drosha antibody

Cells were maintained with DMEM supplemented with 10% FBS and 100 U mL⁻¹ penicillin/streptomycin at 37 °C and 5% CO₂. Ten million cells were collected and lysed in 1 mL non-denaturing lysis buffer at pH 8.0, containing 20 mM Tris-HCl, 125 mM NaCl, 1% NP-40, and 2 mM EDTA supplemented with complete protease inhibitor cocktail. Extracted proteins were incubated overnight with DROSHA antibody (Bethyl Laboratories, A301-886A) at 4 °C; precipitation of the immune complexes was performed with Dynabeads Protein G (Thermo Fisher Scientific, 1003D) for 4 h at 4 °C, according to the manufacturer’s instructions. After immunoprecipitation, the beads were washed three times with the lysis buffer at 4 °C and eluted from the Dynabeads using elute buffer (0.2 M glycine, at pH 2.8). Twenty microliters were loaded onto the gel and the samples were processed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot. The following antibodies were used for the Western blots: ADAR1 antibody (Santa Cruz, sc-73408) and DROSHA antibody (Bethyl Laboratories, A301-886A). The HRP-linked secondary antibodies were used and the blots were visualized with the ECL kit (GE, RPN2232).