Protein g sepharose beads

PANC-1 cells were seeded in 15 cm dishes (5 × 10⁶ cells/dish; 3 dishes were used for each antibody tested) and transfected with 0.5 nM of pre-NC, pre-miR-100 and pre-miR-125b for 24 h. AGO2-RIP was carried out as previously described by our group^{48 (link)}. Briefly, following transfection cells were washed in cold PBS, scraped in PBS and collected by centrifugation. Pellets were then resuspended in lysis buffer (20 mM Tris-HCl pH7.5, 150 mM KCl, 0.5% NP40, 2 mM EDTA, 1 mM NaF, 0.5 mM DTT, 160 U ml⁻¹ RNAsin and protease and phosphates inhibitors) and pre-cleared with Protein G sepharose beads (Sigma) for 2 h at 4°C. Part of cleared lysates (10%) was used as input and the remainder were incubated with Protein G sepharose beads conjugated with anti-AGO2 (11A9, SAB4200085, Sigma-Aldrich) or anti-IgG (Sigma-Aldrich) for 4 h at 4°C. After washing, 10 μl of the immunoprecipitate was kept for western blot analysis and the remainder was treated with DNAse I and proteinase K for 20 min at room temperature. RNA was extracted using phenol/ chloroform and and ethanol/sodium acetate precipitation. RNA was then quantified using Nanodrop.

U251 cells (1 × 10⁶), seeded the day before into 10 cm dishes, were infected with PRVABC59 strain of ZIKV (MOI = 1). At 48 h post-infection, cells were washed three times with PBS before lysing with NP-40 lysis buffer (150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol) containing CompleteTM protease inhibitors (Roche, Mannheim, Germany) on ice for 30 min. Lysates were clarified at 16,000 g for 20 min in a microcentrifuge at 4 °C. Small aliquots of the clarified lysates were kept for loading controls. The remaining lysates were treated with 20 μg/mL RNase A (Roche; Mannheim, Germany) for 1 h on ice, precleared with protein G-Sepharose beads (Sigma Aldrich; St. Louis, MO, USA) for 1 h at 4 °C before sequential incubation with antibodies overnight and then protein G-Sepharose beads for 2 h at 4 °C. Rabbit IgG was used in parallel as a negative control. Immunoprecipitates were washed three times with NP-40 lysis buffer before the bound proteins were eluted by boiling in SDS Sample buffer. Proteins were separated by SDS-PAGE and transferred to (PVDF) membranes for immunoblotting.

3T3-L1 cells were lysed in M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific), mouse tissues were lysed in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) as per the manufacturer's instructions, supplemented with protease and phosphatase inhibitors (Roche) and 5 mM MgCl₂ (Sigma-Aldrich). Protein G Sepharose beads (Sigma-Aldrich) were washed by 1 ml lysis buffer and centrifuged at 1000 g for 1 min at 4°C. Cell or tissue lysates with the pre-washed beads were then incubated for 1 h at 4°C followed by centrifugation at 1000 g for 1 min at 4°C to remove the beads. Then antibody was added to the sample lysate. The antibody-containing pre-cleaned lysate was incubated on a tube rotator overnight at 4°C. After overnight incubation, freshly pre-washed Protein G Sepharose beads (Sigma-Aldrich) were added to the antibody-containing pre-cleaned lysate and incubated for 1 h at 4°C. Then, beads were collected by centrifugation at 1000 g for 1 min at 4°C followed by washing the immune complexes six times using PBS supplemented with protease and phosphatase inhibitors to remove nonspecific binding. The protein–protein interacting partners were then eluted by boiling at 95°C for 5 min. Samples were then directly analysed by SDS-PAGE or stored at −20°C for subsequent use.