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Blood, Vol. 104, Issue 1, 51-57, July 1, 2004
Activation of the MAP kinase pathway by c-Kit is PI-3 kinase dependent in hematopoietic progenitor/stem cell lines
Blood Wandzioch et al.
104: 51
Supplemental materials for: Wandzioch et al, Vol 104, Issue 1, 51-57
Files in this Data Supplement:
- Figure 1. Effect of SF induced stimulation of HPC line on Ras and upstream signaling components (JPG, 132 KB)
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(A) Effect of SF (50ng/mL) stimulation on Shc phosphorylation. Tyrosine phosphorylation of Shc was examined in SF-stimulated HPCs for various times, as indicated (lanes 1-8). HPCs were pretreated with either LY294002 (lanes 5 and 7) or PD98059 (lanes 6 and 8), as indicated (+) for 30 minutes. Lysates were immunoprecipitated with anti-Shc antibodies and immunoblotted with anti-phosphotyrosine antibody PY100 ( -PY). (B) Immunoprecipitation with anti-Shc antibodies, as in panel A, followed by immunoblotted with Grb2 antibodies. (C) Control immunoblot analysis of lysates with anti-Shc antibody. (D) Ras activation assay, precipitation of Ras-GTP with GST-Raf-RBD (all lanes). Ras proteins were detected by immunoblotting with anti-Ras antibodies.
- Figure 2. Analysis of expression levels of c-Kit in different hematopoietic cell populations. (JPG, 62 KB)
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c-Kit expression was analyzed by flow cytometry (A) or real-time polymerase chain reaction (PCR) (B). The analyzed cell populations are the hematopoietic progenitor cell (HPC) line derived from ES cells differentiated in vitro (ES-HPC), myeloid cells generated from the ES-HPC line (ES-HPC-myeloid), and mast cells generated from the ES-HPC lines (ES-HPC-mast). The average fluorescence intensity (M) for the bulk of the cells of the analyzed cell populations is indicated in each histogram (A). The difference in c-Kit levels between the different hematopoietic populations observed by flow cytometry was confirmed on the transcriptional level by real-time PCR using primers specific for the c-Kit receptor (B). The expression level of c-Kit in the different cell populations is in relation to a control gene ( -actin) and was arbitrarily set as 1 in the ES-HPC line.
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