Anti cd98

A total of 293 T cells were transfected with CD98 (OriGene, Rockville, MD; RC200178) using Lipofectamine 3000. Cells were incubated for 48 hours and treated with 100nM Env protein for 1 hour at 37°C. One million cells/immunoprecipitation were lysed using radioimmunoprecipitation assay buffer and immunoprecipitated overnight at 4°C using anti‐IgG (Cell Signaling Technology, Danvers, MA; 5412S), anti‐6XHis (Abcam, Cambridge, UK; 9108), or anti‐CD98 (Invitrogen, PA5‐81032). Complexes were precipitated using Protein A/G beads (Pierce, Rockford, IL) and washed 2× with TNE300 + 0.1% NP‐40, 2× with TNE50 + 0.1% NP‐40, and 2× with PBS + 0.05% Triton X‐100 (PBS‐T). Beads were resuspended in 30μl Tris‐Glycine Sample Buffer supplemented with 2‐mercaptoethanol, incubated at 95°C for 1 hour, and run on a 4 to 20% Tris‐Glycine gel (Invitrogen). The gel was transferred on to a polyvinylidene difluoride membrane using an iBlot2 (Thermo Fisher Scientific, IB24001), blocked for 1 hour with 5% milk in PBS‐T, then incubated overnight with anti‐CD98 (Abcam, 108300). The membrane was incubated with appropriate secondary antibody for 1 hour at room temperature and visualized.