Complete absence of SCL protein in Cre;Scl^fl/fl MKs. (A) Analysis of Scl mRNA levels in hematopoietic cells. RNA was isolated from wild-type C57Bl/6 bone marrow progenitor populations (CMP, MEP, GMP, and MkP) as well as from mature hematopoietic cells (Ter119⁺ splenocytes; bone marrow MKs cultured in vitro for the indicated number of days D1-D3). Quantitative reverse transcription PCR was performed using Scl-specific primers. The y-axis represents the enrichment in cDNA sequences normalized to Gapdh gene control sequences. (B) Analysis of PF4-Cre–mediated excision of Scl floxed alleles in hematopoietic progenitor compartments. (Top) PCR reactions showing amplification of floxed, wild-type (fl and wt, top panel), and excised (Δ, bottom panel) alleles from material isolated from CMP, MEP, GMP, and MkPs purified from Cre;Scl^wt/wt, Cre;Scl^wt/fl, and Cre;Scl^fl/fl bone marrows, as indicated. (Bottom) Semiquantitative PCR reaction measuring the percentage of excision of Scl floxed alleles in Cre;Scl^fl/fl progenitors (supplemental Methods). Data are the mean ± SD of 2 independent experiments. (C) SCL protein is not expressed in Cre;Scl^fl/fl MKs. (Top) Southern blot analysis of genomic DNA isolated from purified Cre;Scl^fl/fl hematopoietic cells. Gra indicates granulocytes; Ery, erythroid cells; and Mac, macrophages. Percentages of excision of the Scl floxed alleles are indicated. MKs from Cre;Scl^wt/wt mice served as controls. (Bottom) Nuclear extracts from hematopoietic cells derived from Cre;Scl^wt/wt, Cre;Scl^wt/fl, and Cre;Scl^fl/fl were analyzed by Western blotting for SCL expression. P300 was used as loading control. Vertical lines have been inserted to indicate repositioned gel lanes.