Anti dcr 2

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)].

Liver tissues were homogenized and digested in 1× RIPA buffer containing phosphatase inhibitor cocktail solution (GenDEPOT, Barker, TX, USA). Western blot experiments were performed following the standard protocol. The following primary antibodies were used: anti-Pan-RAS (sc-14022; Santa Cruz Biotechnology), anti-phospho-AKT (#4060, Cell Signaling Technology), anti-phospho-MEK (#9154, Cell Signaling Technology), anti-phospho-ERK (#4370, Cell Signaling Technology), anti-p21^Cip1 (ab2961; Abcam), anti-p27^Kip1 (ab7961; Abcam), anti-p16^INK4A (10883-1-AP; Proteintech), anti-HP1γ (ab10480, Abcam), anti-DcR2 (ab2019; Abcam), and anti-GAPDH (#2118; Cell Signaling Technology). Anti-rabbit IgG–HRP (Sigma-Aldrich) was used as the secondary antibody. Bands were detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).