SHIP1 expression in NK cells. (A-B) CD56^bright (lanes 1, 3, and 5) and CD56^dim (lanes 2, 4, and 6) primary human NK cells were FACS purified from 3 separate donors. Cell pellets were collected and analyzed for SHIP1 transcript by RT-PCR with 18S rRNA used for nomalization (A) or directly lysed in Laemmli buffer and analyzed for protein by Western blot (B). (B) Western blot was performed using anti-SHIP1 (top panels), anti-SHP1 (middle panels), and anti-GRB2 (bottom panels) Abs sequentially on the same filter. (C-D) NK92 and enriched primary human NK cells were costimulated with monokines IL-12 (10 ng/mL) and IL-18 (100 ng/mL) for 24 hours (panel C, lanes 1-6; panel D, lanes 1-4) or 48 hours (panel D, lanes 5 and 6), after which cell pellets were collected and analyzed for SHIP1 transcript by RT-PCR (C) and for protein by Western blot (D). (D) Western blot was performed with anti-SHIP1 (top panels), anti-SHP1 (middle panels), and antiactin (bottom panels) Abs sequentially in the same filter.