Pbx1 is present throughout B-cell development. (A) FACS-purified CLPs (Lin−CD127intc-kitintSca-1+) were analyzed by immunofluorescence using a Pbx1b-specific monoclonal antibody (left panel). Red staining (Texas red) that was confined to the nucleus (blue staining right panel, DAPI) indicates the presence of Pbx1b. (B) Western blot analysis of double-sorted B-cell populations shows presence of Pbx1b protein in pro-B (B220+CD43+), pre-B (B220+CD43−), and mature B (B220+IgM+) cells that progressively decreases with increasing maturation. Pbx1+/+ and Pbx1−/− MEFs were used as positive and negative controls, respectively. Faint cross-reactive band in last lane is not representative of residual Pbx1b protein. (C) Pbx1 transcripts were assessed in purified progenitors and B-cell populations by quantitative RT-PCR analysis. Relative levels of expression are compared to long-term HSCs. Error bars represent SEMs for triplicate analyses.
