EWI-2 promotes increased CD81-α4β1 complex formation. (A) MOLT-4 cells were biotinylated and lysed in 1% Brij 58 (lanes 1-6) or in 1% Brij 58 followed by preclearing and addition of 1% Triton X-100 (lanes 7-12). Lanes 4′ to 6′ are longer esposures of lanes 4 to 6. Proteins were immunoprecipitated as indicated and visualized with ExtrAvidin-HRP (Sigma). Note that CD81 is not detected in Figure 5A, lanes 3 and 4, because associated proteins shield it from biotin labeling. The background band of approximately 50 kDa precipitated with EWI-2 (lane 5′) is not MHC class I because it does not comigrate in higher resolution SDS-PAGE (not shown). Numbers to the right of panel A indicate the sizes of molecular weight markers (in kDa). (B-C) MOLT-4 cells were surface biotinylated and lysed in 1% Brij 58 (lanes 1-8) or Brij 58 followed by Triton X-100 (lanes 9-12). The indicated proteins were immunoprecipitated using the following antibodies: W6/32 (MHC I), A4PUJ (α4), TS2/16 (β1), M38 (CD81), and polyclonal anti–EWI-2 antiserum. CD81 (panel B) and α4β1 (panel C) were visualized by ExtrAvidin-HRP. (D) MOLT-4 cells were biotinylated and lysed in 1% Brij 58 or in 1% Brij 58 followed by preclearing and addition of 1% Triton X-100 (TX100). Proteins were immunoprecipitated with either anti-CD81 (M38, JS64) or anti–HLA I (W6/32) mAbs.