Figure 4

Nef binds AIP1 in vitro and invivo.(A)Nef binds AIP1 in vitro . GST and GST.AIP1 fusion proteins were expressed in E. coli and purified by glutathione S-transferase beads. They were incubated with V5 epitope-tagged wild type Nef and mutant NefΔ YPL proteins expressed in IVT. Bound proteins were resolved by 10% SDS-PAGE followed by western blotting with α V5 antibodies. GST was used as the negative control (top right panel, lane 2). 10% of input proteins (inputs) is presented to the left of GST pulldowns. (B) Nef binds AIP1 in cells. HA epitope-tagged AIP1 protein was expressed alone or with the wild type and mutant NefYPL proteins in 293T cells. Cells were disrupted by dounce homogenization in hypotonic buffer containing protease inhibitor cocktails, followed by incubation with α HA polyclonal antibodies and protein-G beads. After the immunoprecipitation, western blotting was performed using α Nef antibodies (top left panel). A control western blot for 10% of input proteins was performed with α Nef and α AIP1 antibodies (bottom left panels).