Gsh agarose beads

Cells were solubilized with 1% CHAPS lysis buffer [50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA supplied with protease inhibitor cocktail (Roche)]. Lysates were clarified by centrifugation at 12,000 ×g for 5 min, and the supernatants were incubated with antibodies with gentle rocking at 4°C overnight. Protein A or G agarose beads (GE Healthcare) were pre-equilibrated with ice-cold lysis buffer, applied to the protein–antibody mixture, and incubated for another 1–2 h at 4°C. For immunoprecipitation, mouse IgG2a (M9144, Sigma-Aldrich) or IgG1 (M3198, Sigma-Aldrich) was used as a control. The beads were then centrifuged down and washed thrice with the lysis buffer. For GST pulldown assays, cells were lysed in 1% CHAPS lysis buffer on ice for 30 min. Lysates or synthetic Aβ40 diluted with lysis buffer were clarified by centrifugation for 5 min at 12,000 × g, and incubated with GST-fusion proteins pre-bound to GSH-agarose beads (GE Healthcare) for 2 h at 4°C. The precipitates were solubilized in Laemmli sample buffer and subjected to immunoblotting analysis.

MDA-MB-231 cells were cotransfected with pcDNA3.1-MS2, pcDNA3.1-MS2- NUDT3-AS4, pcDNA3.1-MS2- NUDT3-AS4 –Mut (miR-99s) and pMS2-GST. After 36 h, cells were used to perform RNA immunoprecipitation (RIP) experiments and RNA purification using GSH agarose beads (GE Healthcare) and the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA) as described previously^{22 (link)}. pMS2-GST and pMS2 plasmid were gifts from Dr. Gorospe^{22 (link)}. The RNA fraction isolated by RIP was quantified by NanoDrop ND1000 (Thermo-Fisher Scientific, Waltham, MA) and its quality was assessed by Agilent 2100 Bioanalyzer (Agilent).