Immunoprecipitation and Nuclease Assays

Immunoprecipitation of HA-tagged proteins was performed as described [20 (link)]. Immunoprecipitation of endogenous proteins was performed as described [18 (link)] using 100 μg of crude nuclear or 175 μg refined cytoplasmic or nuclear extracts. S1 nuclease protection assay was performed as described [20 (link)]. Monoclonal and polyclonal HA antibodies (Cat#s MMS-101R and PRB-101C, respectively, Covance) were used for both IP (3 μL) and WB (1:1000). Anti-CPSF73, anti-Symplekin, and anti-CPSF100 antibodies (1:1000) were described previously [18 (link), 26 (link)]. Commercial anti-Dcr-2 (Abcam ab4732), anti-Actin (Abcam ab8227), anti-H3 (Cell Signaling 4499), and anti-MEK1/2 (Cell Signaling 8727) were used at manufacturer recommended concentrations. The anti-R2D2 antibody was a generous gift from the Siomi lab [27 (link)].