Analysis of s-SHIP and SHIP mRNA expression in ES cells and hematopoiesis.

(A) Northern Blot analysis of SHIP mRNA species expressed by ES cells (TL1) and lineage-committed hematopoietic cell lines (70Z/3, A20, BAL17). A full-length SHIP cDNA clone was used to probe the filter. After initial hybridization with the SHIP probe, the filter was stripped and reprobed for β-actin to confirm that comparable amounts of RNA were loaded in each lane. (B) RT-PCR analysis of s-SHIP expression in ES cells (TL1, E10) and lineage-committed hematopoietic cell lines (70Z/3, WEHI-231). The primer pair ESHIP23 and ASHIP1098 selectively amplifies s-SHIP mRNA and not SHIP mRNA. Analysis of the hepatoma cell line, Hepa 1-6, serves as a negative control for SHIP. (C) RPA analysis of s-SHIP and SHIP expression in ES cells and hematopoietic development. After annealing and RNase digestion, the s-SHIP/SHIP probe is protected for 247 nucleotides by s-SHIP mRNA and for 203 nucleotides by SHIP mRNA. Thus, a single probe is used to detect the relative abundance of the s-SHIP and SHIP mRNAs. Each RNA sample was incubated with a GAPDH probe as an internal control. Analysis of NIH3T3 cell RNA is provided as a negative control for both SHIP and s-SHIP mRNA expression.