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Blood, Vol. 109, Issue 9, 3945-3952, May 1, 2007
K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells
Blood Van Meter et al.
109: 3945
Supplemental materials for: Van Meter et al, Vol 109, Issue 9, 3945-3952
Files in this Data Supplement:
- Figure S1. Validation of FACS-based phospho-protein analysis in a uniform population of cells (PDF, 457 KB) -
(A) Ba/F3 murine pro-B cells were cytokine starved for 6 hours prior to stimulation with IL-3 (10 ng/mL) for 5 to 60 minutes. Cells were split and levels of pERK and pSTAT5 were analyzed by Western blotting and flow cytometry. (B) Ba/F3 cells were cytokine starved for 16 hours prior to stimulation with IL-3 (10 ng/mL) for 10 to 60 minutes. Cells were split and levels of pS6 were analyzed by Western blotting and flow cytometry.
- Figure S2. Validation of FACS-based phospho-protein analysis in primary c-kit+ lin–/lowcells (PDF, 939 KB) -
Bone marrow harvested from femora, tibiae, and humeri of 6 wild-type (WT) mice was pooled and depleted of lin+ cells using a magnetic lineage-cell depletion kit (anti-CD5, anti-B220, anti-CD11b, anti–Gr-1, anti-7/4, and anti–TER-119; Miltenyi Biotec, Auburn, CA) following the manufacturer’s instructions. Labeled cells were divided into linhi and lin–/lowfractions with an AutoMACS separator (Miltenyi Biotec) running the DEPLETE program. The lin–/low fraction was then subfractionated using anti–c-kit magnetic beads (Miltenyi Biotec) and the POSSEL AutoMACS program. Each of the 3 fractions was starved, stimulated with GM-CSF 10 ng/mL, then analyzed by immunoblotting and FACS as described in “Materials and methods.” (A) Staining of c-kit versus lineage markers in cells before sorting (top panel) and in each fraction after magnetic sorting (middle panel). Lower panels show levels of pErk and pS6 measured by Western blot and FACS in the sorted fractions. (B) Levels of pErk and pS6 measured by FACS, with gating on the targeted population subsets for further enrichment. The data demonstrate agreement between immuoblotting and FACS analysis and show that FACS provides superior isolation of targeted populations.
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