Figure 2

p37-GFP and TIP47 co-immunoprecipitate in the presence of non-ionic detergent. A. Membrane fractions from BSC-40 cells infected with vvF13LGFP at an MOI of 5 were extracted as described in Materials and Methods. Protein extracts obtained from the high speed pellet were resuspended in a buffer containing a non-ionic detergent (0.5% NP-40, 150 mM NaCl, 20 mM Tris, pH7.4) and immunoprecipitated with antibodies specific for TIP47 or GFP followed by SDS-PAGE analysis and immunoblot. Antibody raised against the trans Golgi marker, TGN46 (Sigma Aldrich) was used as a non-specific control for immunoprecipitation. Lane 1, IP using TIP47 pab; Lane 2, Unbound Fraction; Lane 3, IP using TGN46 pab; Lane 4, Unbound Fraction; Lane 5, IP using GFP pab, Lane 6, Unbound Fraction, Lane 7, IP using TGN46 pab; Lane 8, Unbound Fraction; B. Immunoblot of whole cell extracts and membrane fractions immunoprecipitated with GFP pab from BSC-40 cells mock-infected or infected with vvWR, vvGFP, vvΔF13LGFP, or vvF13LGFP at an MOI of 5 probed with GFP mab. Lane 1 and 6, mock-infected, Lane 2 and 7, vvWR, Lane 3 and 8, vvF13LGFP, Lane 4 and 9, vvΔF13LGFP, Lane 5 and 10, vvGFP. IP, immunoprecipitated; pab, polyclonal antibody; mab, monoclonal antibody; ** pGFP; * p37-GFP fusion protein; † TIP47 protein.