Identification of EWI-2 as a major α4β1-associated protein. (A) Coomassie blue staining of proteins eluted from anti-α4 antibody beads with Triton X-100 SDS (lane 1) and remaining on the beads (lane 2). The sharp band of approximately 110 kDa corresponds to nonspecifically associated nucleolin. (B) EWI-2 coimmunoprecipitates with α4β1 and CD81. CD81 (mAb M38, lanes 1 and 4), α4β1 (A4PUJ, lanes 2 and 5), and MHC class I (W6/32, lanes 3 and 6) were immunoprecipitated from 1% Brij 58 lysate of MOLT-4 cells stably expressing either EWI-2–FLAG or vector control. EWI-2 protein was visualized using biotinylated anti-FLAG mAb M2. (C) MOLT-4 cells stably transduced with EWI-2–flag construct were cell-surface biotinylated and lysed in 1% Brij 58. Lysate from 107 cells was centrifuged at 15 000g for 15 minutes. The resulting supernatant was used for immunoprecipitation (lanes 1 and 2), or further centrifuged for 45 minutes at 100 000g to yield 100 K supernatant (lanes 3 and 4) and 100 K pellet that was resuspended in 1% Triton X-100, 0.1% SDS (lanes 5 and 6). Immunoprecipitations with anti-CD81 (mAb M38) or anti–EWI-2 pAb were as indicated. Note that anti–EWI-2 pAb recognizes 2 forms of EWI-2 on the cell surface, whereas only one (mature) form of EWI-2 associates with CD81. Numbers to the left (panel A) and right (panels B-C) of the blots indicate the sizes of the molecular weight markers (in kDa).
