Normal mouse igg beads

Cells were seeded at a density of 1.2×10⁶ cells/900μL of a 6-well plate (BD Biosciences, 353046) (3 wells were used per each cell line tested) and incubated with 0.66 μM ³H-Ribavirin for 8hrs at 37°C, 5% CO₂. Following incubation, cells were washed three times with PBS and scraped in IP buffer (50 mM Tris pH 5.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP40 and 1% Triton X-100). Cells were then homogenized using a glass Dounce homogenizer and left to rotate at 4°C for 30 minutes. After centrifugation at 10,000 rpm for 10 minutes at 4°C, supernatants were split into two tubes and pre-cleared with normal mouse-IgG beads (Santa Cruz, sc-2343) for 30 minutes at 4°C. After spinning at 500×g for 5 minutes, protein concentrations were quantified using Pierce BCA Protein assay (Thermo Scientific, 23223). 100–200 μg of protein lysates were used for immunoprecipitation with 8μg of normal mouse IgG (Millipore, 12-371) or mAb mouse anti-eIF4E (P-2) (Santa Cruz, sc-9976) antibodies coupled to Protein A/G PLUS-Agarose (Santa Cruz, sc-2003). Immunoprecipitations were carried out overnight at 4°C. Beads were washed 6 times with IP buffer and eluted with 80 μl 2× Laemmli sample buffer (BioRad) for 15 minutes at 99 °C. After spinning beads supernatants were used for scintillation counting and western analysis (probed with rabbit anti-eIF4E antibody).