SHIP protein expression is up-regulated in CD34⁺ CML cells. (A) The top panel shows an example of real-time RT-PCR SYBR Green1 fluorescence versus cycle number of human SHIP and GAPDH cDNAs prepared from the RNA isolated from FACS-sorted CD34⁺lin^–, CD34^–lin⁺, CD34⁺CD38^–lin^–, and CD34⁺CD38⁺lin^– normal BM (n = 4) and CML (n = 8) cells. Bottom panel shows the levels of human SHIP transcripts relative to GAPDH in each population expressed as a percentage of the levels of SHIP transcripts detected in the CD34⁺CD38⁺lin^– subset of normal BM cells. The level of SHIP transcripts in each individual sample was calculated using the formula described.²⁶  (B) The top panel shows a Western blot analysis of cell lysates prepared from one normal BM sample (lanes 1-3) and 3 CML samples (lanes 4-12). The lysates from equal numbers of cells were separated on 8% polyacrylamide gradient gels. The filter was first probed with a human anti-SHIP antibody (P1C1) (upper blot), then stripped and reprobed with an antiactin antibody (lower blot). The percentage of CD34⁺ cells in each population is indicated below each lane. In the lower part of panel B, the measured levels of human SHIP protein relative to actin are shown.