Endosome isolation and Western blot analysis

Purified CD8⁺ T cells were homogenized as previously described (Graham, 2002) in Diluent (50 mM Hepes-NaOH, 500 mM KOAc, and 5 mM MgOAc) and Halt protease and phosphatase inhibitor with a 27G needle 25 times and mixed with 50% solution (Diluent, 0.25 mM sucrose; Optiprep) for a 30% density. The gradient was loaded from bottom to top with the following densities: 2 ml of 30% homogenate, 8 ml of 25%, and 2 ml of 5%. Gradients were subjected to 250,000 g for 3 h at 4°C. Endosomes were collected from the 25–5% interface and processed for immunoprecipitation with anti-GFP (mouse monoclonal 3E6; Molecular Probes) covalently linked to CrossLink IP beads (Pierce). YFP⁻ endosomes were diluted in PBS and subjected to 100,000 g for 10 h at 4°C. Both YFP⁺ beads and YFP⁻ pellet were resuspended in Laemmli sample buffer (Bio-Rad) and boiled. For Western blotting, the membrane was blocked with 5% nonfat milk or 5% BSA in PBS plus 0.1% Tween 20 for 30 min after proteins were transfer from PAGEr Gold Precast 4–20% Tris-Glycine gel (Lonza). Blots were incubated overnight at 4°C with 1:1,000 of anti-GFP (ab290; Abcam), CD3z (H146-968; Thermo) EEA1 (CST), Rab4 (BD), Rab7 (CST), Rab8 (CST), Rab11 (Abcam), Rab13 (Abcam), Rab21 (Santa Cruz), and Rab27a (Santa Cruz). The membrane was then incubated with 1:5,000 horseradish peroxidase–conjugated anti–rabbit or anti–mouse Fc-specific IgG antibody (Jackson ImmunoResearch) for 1 h at room temperature. Protein was detected using Super Signal chemiluminescent reagent (Thermo). Where described, native PAGE Bis-Tris Gels (nonreducing) were used (Thermo).