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Blood, Vol. 114, Issue 10, 2172-2180, September 3, 2009
M-CSF elevates c-Fos and phospho-C/EBP (S21) via ERK whereas G-CSF stimulates SHP2 phosphorylation in marrow progenitors to contribute to myeloid lineage specification
Blood Jack et al.
114: 2172
Supplemental materials for: Jack et al
Files in this Data Supplement:
- Figure S1 (JPG, 647 KB)
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(A) Ba/F3 (MR, GR) cells were cultured in IL3, in M-CSF, G-CSF, IL3, and M-CSF, or IL3 and G-CSF for 3 days or 8 days, and cell morphology was visualized by cytospin followed by Wright’s-Giemsa staining (cells died in M-CSF before day 8). Photomicrographs were taken using a Zeiss Axiophot microscope (Carl Zeiss, Thornwood, NY), a Kontron Electronik Progress 3012 camera (Kontron, Munich, Germany) and a 63×/1.40 NA oil objective. (B) FACS analysis for Mac1 and Gr-1 was carried out on Ba/F3(MR, GR) cells cultured for 48 hrs in either G-CSF or M-CSF and on mouse bone marrow cells (mBM).
- Figure S2 (JPG, 253 KB)
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(A) Protein extracts were prepared from Ba/F3(MR, GR) cells proliferating in IL-3 or after transfer to G-CSF or M-CSF for 3, 24, or 48 hr. These were subjected to Western blotting for PU.1, C/EBPα, and β-actin. (B) Ba/F3 (MR, GR) cells were removed from IL-3 for 1 hr and placed in the indicated doses of M-CSF or G-CSF for 10 min followed by analysis of P-ERK, ERK, and β-actin levels by Western blotting. (C) Ba/F3 (MR, GR) cells were removed from IL-3 for 1 hr and placed in no cytokine, 10 ng/mL G-CSF, 10 ng/mL M-CSF or both G-CSF and M-CSF for 10 min and analyzed for P-ERK, ERK, P-SHP2(Y542), SHP2, P-STAT3, and STAT3.
- Figure S3. Cell extracts were prepared from 293T cells transiently transfected with either pMT2 (−) or with constitutively active pMT2-SHP2(E76K) and from Ba/F3(MR, GR) cells grown in IL-3, in the absence of IL-3 for 3 hrs (C), or in the absence of IL-3 for 3 hrs followed by addition of 10 ng/mL M-CSF (M) or 10 ng/mL G-CSF (G) for 10 min (JPG, 458 KB)
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Cells were lysed in 0.1% Triton-X 100, 20 mM Hepes, pH 7.5, 300 mM NaCl with protease inhibitors (Sigma), but lacking sodium orthovanadate and EDTA, and immunoprecipitated with anti-SHP2 antibody (C-18, Santa Cruz) and protein A-agarose beads. Western blotting confirmed equivalent SHP2 protein content of each immunoprecipitate (not shown). SHP2 activity was assessed using PTP assay kit 1 (Millipore, Billerica, Massachusetts, USA). Immunoprecipitates were washed twice with ice-cold 0.1% Triton-X 100 lysis buffer and then twice with ice-cold kinase buffer (25 mM HEPES, pH 7.2; 50 mM NaCl, 2.5 mM EDTA), and the beads were resuspended in kinase buffer supplemented with 5 mM DTT. 12.5 µl of beads suspension (2E6 cell equivalents) was then incubated with 12.5 µl of 1 mM tyrosine phosphopeptide (Millipore) for 30 minutes at room temperature, and supernatants were transferred to a 96-well plate. Malachite green solution (Millipore) was added and allowed 15 minutes for color development at room temperature. The absorbance was measured on a microplate reader (Bio-Rad, Hercules, CA, USA) at 655 nm. The activity obtained with Ba/F3(MR, GR) cells in IL-3 was set to 1.0, and results from three experiments with Ba/F3(MR, GR) cells is shown (mean and S.E.). (B) Phosphatase activities were obtained similarly using extracts from Ba/F3(MR, GR) cells cultured in IL-3 or starved for IL-3 for 1 hr and transferred to G-CSF for 10 min, with or without addition of 50 µM NSC-87877 for 1 hr prior to extract preparation. (mean and S.E. from three determinations). ** P=0.001 relative to IL-3 without drug; * P=0.05 relative to G-CSF without drug. (C) Western blot analysis for phospho-SHP2(Y542) and total SHP2 in the same lysates used for immunoprecipation.
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