Immunoprecipitation and Oligo(dT) pulldown

One 10 cm dish of wild type HEK293T cells were grown to ~80% confluency for each replicate of each experiment. Cells were scraped from the dish and pipetted with media into a 15 ml conical vial.The cells were pelleted by centrifugation at 300 xG for 2 minutes and media was aspirated and discareded. The cell pellet was then resuspended  2 ml of ice cold PBS, centrifuged at 300 xG for 2 minutes and PBS was aspirated and discarded. This PBS wash was repeated for a total of 2 washes. The cell pellets were then resuspened in 500 µl IP lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5 % Triton X100)  supplemented with 50 mM iodoacetamide, 50 μM PR-619, 0.5 μM bortezomib, and protease and phosphatase inhibitor mini tablets (Pierce). Lysates were vortexed to ensure lysis and then centrifuged for 10 min at 4°C at 20,000 x g. Following centrifugation the supernatant was collected for further analysis: 40 µl was removed and added to 15 µl 1 xLDS sample buffer with 6 µl of Beta mercaptoethanol for the input sample. The remaining ~450 µl was added to 50 µl of Oligo d(T)25 magnetic bead slurry (New England Biolabs) or 50 µl protein G dynabeads pre-conjugated to the indicated antibody. To prepare the protein G Dynabeads (Invitrogen), the slurry was placed in a 1.5 ml tube in a magnetic separating tube rack, the packaging buffer was removed and discarded. The beads were then resuspended in 200 µl PBS with 5 µg of PABPC1 antibody (Abcam, ab21060) and incubated for 10 minutes with rotation at room temperature. Lysates were incubated overnight at 4°C with the magnetic bead resin with constant rotation. Following overnight incubation, the unbound sample was removed using a magnetic separating tube rack. Beads were then resupended with fresh lysis buffer and incubated for 15 min at 4 deg. C. This was repeated twice for a total of 3 washes. Beads were then resuspended in 50 µl 1x LDS sample buffer with reducing agent and incubated at 90 deg C for 1 minute to elute bound proteins. Samples were then stored at -20 deg C until analysis by SDS PAGE and mass spectrometry.