1. Expression MosIR binding by dsRBPs TARBP and PACT.
Expression MosIR binding by dsRBPs TARBP and PACT. (A) Western blot showing expression and immunoprecipitation of HA-tagged TARBP2 and PACT proteins using anti-HA antibody. For TARBP2 and PACT immunoprecipitation, cultured cells were collected into 2 ml of PBS 48 h after transfection. The samples were centrifuged at maximal speed for 1 min, and the supernatants were removed. The cells were lysed with 200 μl of lysis buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 0.5% IGEPAL-25%, 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na3VO4, supplemented with 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]), passed through a needle and stored at ice for 10–15 min. The samples were centrifuged (15 min, 4°C, 12,000 g), and the supernatants were used for immunoprecipitation. The supernatants were diluted with binding buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na3VO4, 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]) to 0.1% total concentration of IGEPAL-25. HA-probe antibody (F-7, Santa Cruz Biotech., sc-7392 AC) conjugated with agarose beads was used for the protein immunoprecipitation. The beads were added to each sample and incubated on rotator overnight at 4°C. The beads were washed with 1,000 μl of binding buffer 5 times (centrifugation at 4°C, 4,000 g, and 4 min). The last wash was placed into a new tube. Immunoprecipitated RNA was quantified using RT-qPCR. (B) Relative enrichment of CAG-EGFP-MosIR RNA upon immunoprecipitation of TARBP2 and PACT.
Tomas Demeter et al. LSA 2019;2:e
© 2019 Demeter et al.