Immunoprecipitation of FLAG-tagged Ago2 from HEK 293 cells

For immunoprecipitation of FLAG-tagged Ago2 from HEK 293 cells, FLAG-M2 Agarose beads (Sigma-Aldrich) were used as described previously (25 (link)). Proteins and RNAs were extracted from beads for further analysis. For Western blotting, proteins were transferred to polyvinylidene fluoride or polyvinylidene difluoride membrane after SDS–PAGE electrophoresis followed by blocking and probing with either of the following primary antibodies (dilution mentioned also) at 4°C overnight. Mouse anti-Ago2 (Abnova) (1:1,000), Rabbit anti-calnexin (Cell Signaling Technology) (1:1,000), Rabbit anti-HRS (Bethyl Laboratories) (1:1,000), Rat anti-HA (Roche) (1:1,000), HRP-conjugated β–Actin (Sigma-Aldrich) (1:10,000), HRP-conjugated GAPDH (Sigma-Aldrich) (1:50,000), Rabbit anti-COX4 (Cell Signaling Technology) (1:1,000), Rabbit-LAMP 1 (Cell Signaling Technology) (1:1,000), Mouse-Alix (SantaCruz) (1:200), Mouse-P-Ago2 (4G10) (Millipore) (1:1,000), and Rabbit anti-S3 (1:1,000) were used with dilutions mentioned here.