1. Activation of JAK2 in Human Vascular Endothelial Cells by Granulocyte-Macrophage Colony-Stimulating Factor
by Raffaella Soldi, Luca Primo, Maria Felice Brizzi, Fiorella Sanavio, Massimo Aglietta, Nadia Polentarutti, Luigi Pegoraro, Alberto Mantovani, and Federico Bussolino
Blood
Volume 89(3):
February 1, 1997
©1997 by American Society of Hematology

2. Cross-linking of [125I]GM-CSF to ECs (A) and U937 cells (B) and displacement by GM-CSF.
Cross-linking of [125I]GM-CSF to ECs (A) and U937 cells (B) and displacement by GM-CSF. Quiescent ECs at passage II and U937 cells were incubated in binding medium with 0.5 nmol/L [125I]GM-CSF without or with 100-fold excess of unlabeled ligand in the presence of 1 mmol/L disuccinyl suberate. Proteins were separated by SDS-PAGE and visualized by autoradiography. Three experiments with ECs and 2 with U937 cells have been performed with similar results.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology

3. RT-PCR analysis of expression of α chain of GM-CSFR in ECs (lanes 3 to 5) and U937 cell line (lanes 7 to 9).
RT-PCR analysis of expression of α chain of GM-CSFR in ECs (lanes 3 to 5) and U937 cell line (lanes 7 to 9). One microgram of total RNA was reverse-transcripted and amplified with GM-CSFRα1 –(lanes 4 and 8), GM-CSFRα2– (lanes 5 and 9), and β-actin– (lanes 3 and 7) specific oligonucleotides. Amplified products were run in an ethidium bromide-agarose gel and visualized by UV. In lane 1, molecular weight have been runned. Lanes 2 and 6 represent two control experiments performed with RT-PCR mixture without cell RNA. This experiment is representative of five performed with similar results.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology

4. Northern blot analysis of α chain of GM-CSFR and of β actin in ECs and in U937 cell lines.
Northern blot analysis of α chain of GM-CSFR and of β actin in ECs and in U937 cell lines. Seven micrograms of PolyA+ RNA was run in a formaldehyde-agarose gel and after blotting to Duralon membrane, hybridized to α-chain– and β-actin–specific probes. Transcripts have been visualized by autoradiography. This experiment is representative of three performed with similar results.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology

5. Time course of GM-CSF–induced tyrosine phosphorylation of JAK2.
Time course of GM-CSF–induced tyrosine phosphorylation of JAK2. Quiescent ECs were incubated with 10 ng/mL of GM-CSF in M199-BSA at 37°C, lysed, and proteins immunoprecipitated with anti-JAK2 antibody. Immunoprecipitate was analyzed by SDS-PAGE followed by immunoblotting with antiphosphotyrosine antibody. Subsequently, blots were reprobed with anti-JAK2 antibody (lower panel). This experiment is representative of five performed with similar results.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology

6. Time course of GM-CSF–induced tyrosine phosphorylation of JAK1.
Time course of GM-CSF–induced tyrosine phosphorylation of JAK1. Quiescent ECs were incubated with 10 ng/mL of GM-CSF in M199-BSA at 37°C, lysed, and proteins immunoprecipitated with anti-JAK2 antibody. Immunoprecipitate was analyzed by SDS-PAGE followed by immunoblotting with antiphosphotyrosine antibody. Subsequently, blots were reprobed with anti-JAK1 antibody (lower panel). This experiment is representative of two performed with similar results.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology

7. Dose dependence of GM-CSF–induced tyrosine phosphorylation of JAK2.
Dose dependence of GM-CSF–induced tyrosine phosphorylation of JAK2. Quiescent ECs were incubated with different amounts of GM-CSF (ng/mL) in M199-BSA for 5 minutes at 37°C, lysed, and proteins immunoprecipitated with anti-JAK2 antibody. Immunoprecipitate was analyzed by SDS-PAGE followed by immunoblotting with antiphosphotyrosine antibody. Subsequently, blots were re-probed with anti-JAK2 antibody (lower panel). This experiment is representation of five performed with similar antibody. In this experiment, the 200-kD molecular marker remained at the top of the gel.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology

8. In vitro phosphorylation of VLPQDKEYYKVKEPGE substrate.
In vitro phosphorylation of VLPQDKEYYKVKEPGE substrate. (A) Dose-dependent effect of GM-CSF–induced activation of JAK2 in ECs (•) and U937 cells (⋄). Cells were stimulated for 5 minutes with increasing amount of cytokine. Anti-JAK2 immune-complexes (•, ⋄) or immune complexes obtained with nonimmune rabbit serum (○), in triplicate, were incubated in kinase buffer containing 5 μg of peptide and processed as described in Materials and Methods. (B) Time course of GM-CSF (50 ng/mL)–dependent activation of JAK2 in ECs. (C) Effect of stimulation of JAK2 catalytic activity in ECs treated for 5 minutes with GM-CSF (50 ng/mL), heat-inactivated GM-CSF (50 ng/mL), GM-CSF (50 ng/mL) neutralized by specific antibody or by nonimmune serum, and IL-5 (50 ng/mL). Mean ± SD of three samples in one experiment representative of three performed.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology

9. GM-CSF–induced tyrosine phosphorylation of JAK2 associated with GM-CSFR β chain.
GM-CSF–induced tyrosine phosphorylation of JAK2 associated with GM-CSFR β chain. Quiescent ECs were incubated for 5 minutes at 37°C in M199-BSA with GM-CSF (50 μg/mL) and cell lysate were immunoprecipitated with β-chain antiserum. After protein solubilization from Protein A-Sepharose, samples were further subjected to a second immunoprecipitation with anti-JAK antibody. Samples were analyzed by SDS-PAGE followed by immunoblotting with antiphosphotyrosine antibody. Subsequently, blot was reprobed with anti-JAK2 antibody (lower panel). The figure is representative of five similar experiments.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology

10. Time course of GM-CSF–induced tyrosine phosphorylation of GM-CSFR β chain.
Time course of GM-CSF–induced tyrosine phosphorylation of GM-CSFR β chain. Quiescent ECs were incubated with GM-CSF (20 ng/mL) in M199-BSA at 4°C and cell lysates were immunoprecipitated with β-chain antiserum. Immunoprecipitate was analyzed by SDS-PAGE, followed by immunoblotting with antiphosphotyrosine antibody and then with anti–β-chain antibody. The figure is representative of two identical experiments.
Raffaella Soldi et al. Blood 1997;89:
©1997 by American Society of Hematology