BCAP is expressed in human and murine NK cells, recruits PI3-K upon tyrosine phosphorylation, and is involved in Akt signaling. (A) Whole cell lysates from (0.5 million cells/lane) were separated by SDS-PAGE and immunoblotted for BCAP. Multiple bands are the result of alternative splicing,²²  and the weak band near 85 kDa was nonspecific in all lysates tested. (B) NK-92 cells (40 million/sample) were treated with (+) or without (−) pervanadate for 10 minutes at 37°C, lysed, immunoprecipitated with anti-MHC-I mAb (W6/32) and anti-BCAP mAb (4L8E6), separated by SDS-PAGE, and immunoblotted for phosphotyrosine, BCAP, and the p85 subunit of PI3-K. (C) NK cells from wild-type (WT) and BCAP^−/− mice were MACS purified and cultured with IL-2 for 1 week. Whole cell lysates (10⁶/sample) were separated by SDS-PAGE and immunoblotted for BCAP and GAPDH. (D) Purified NK cells from WT or BCAP^−/− mice were sorted and cultured in IL-2 for 8 days, then stimulated with biotinylated anti-NK1.1 antibody and streptavidin. Cells were lysed at the indicated times after streptavidin addition, and Akt phosphorylation was resolved by Western blot. Immunoblotting for GAPDH levels was performed as a loading control. No streptavidin was added to the time 0 samples, and the positive control was treated with pervanadate for 10 minutes. The result is representative of 4 separate experiments.