Intracellular LFA-1 contained in Rab27 endosomes. (A) Schematic depicting the mYFP tag on the exterior of LFA-1⁺ endosomes. (B) Purity of endosomal fraction isolated from naive CD11a-mYFP/CD8⁺ T cell homogenate was confirmed by the absence of lamin B (nucleus marker), HSP90 (cytosol maker), NaKATPase (plasma membrane [PM] marker), and SERCA1/2/3 (ER–Golgi marker) signals and the presence of CD11a-mYFP signal in Western blot analysis (left). LFA-1⁺ endosome and LFA-1⁻ endosome were further separated by immunoprecipitation (IP) with a monoclonal GFP/YFP (E36) antibody and tested for the presence of CD3ζ (right). Results are representative of three independent experiments. (C) Total cell lysate (TCL) and LFA-1⁺ endosome lysate (Endo) were isolated under native conditions (native) and tested for the presence of intact LFA-1 heterodimer, and compared with endosomes and TCL isolated under denaturing conditions (denatured) for the presence of CD11a-mYFP (180 kD) and CD18 (∼95 kD). Results are representative of three independent experiments. (D) LFA-1⁺ and LFA-1⁻ endosomes were tested for indicated Rab protein expression. Results are representative of six independent experiments.