Proteinase cocktail inhibitors edta free tablets

Flag Co-IP assays were carried out as previously described ⁵⁹ . Briefly, 293T cells were seeded at 4 × 10⁶ cells per 10-cm dish and transfected the next day using standard methods. The following DNA amounts were used to transfect 293T cells: 4 μg Flag-A3F, 4 μg CBFβ-myc, and 2 μg Vif-HA expression plasmids. Next day, the cells were treated with MLN-4924 (BioVision; Cat # 2566) at 2 μM final concentration for 24 hours to prevent Vif-mediated proteasomal degradation of A3F. 48 hours post-transfection, total cell lysates were harvested in 1 mL of RIPA lysis buffer (Sigma) supplemented with proteinase cocktail inhibitors EDTA-free tablets (Roche) with or without benzonase® nuclease (250U/ml final concentration; Sigma) addition and Flag Co-IP was performed at 4°C for overnight. To detect eluted complexes as well as the input cell lysates, western blotting was performed. CBFβ was detected using a rabbit anti-myc polyclonal antibody at a 1:5000 dilution, Vif was detected using a rabbit anti-HA monoclonal antibody at a 1:5000 dilution, and A3F detected using a mouse anti-Flag antibody at a 1:5000 dilution. HSP90 was used as loading control and detected with mouse anti-HSP90 antibody at a 1:20000 dilution. Protein bands were visualized using an Odyssey® Infrared Imaging System (Licor) as described above.