Expression pattern of the various SHIP constructs in SHIP^−/− BMMCs.

Uninfected SHIP^−/− and SHIP^+/+ BMMCs are shown in the first 2 panels/lanes in each figure. (A) FACS analysis of the BMMCs expressing the various SHIP constructs following 2 months in suspension culture. GFP was used as a marker for SHIP protein expression (top panel) and c-kit (using PE-tagged anti-c-kit) for SF receptor levels (bottom panel). (B) Northern blot analysis of the BMMCs expressing the various SHIP constructs following 2 months in suspension culture. SHIP messenger RNA (mRNA) levels were determined using a 250-bp fragment of the most 5′ part of the first exon of SHIP as a probe (top panel). The blot was reprobed with the entire puromycin cDNA removed from the MSCVpac viral vector as an independent measure of infection (bottom panel). The last lane (R34G) was from a 4-month suspension culture. (C) Western blot analysis of total cell lysates from SDS-sample buffer lysed BMMCs expressing the various SHIP constructs following 2 months in suspension culture to measure SHIP protein levels using anti-SHIP antibodies.