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First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02740

Journal of Cell Science 119, 380-387 (2006)
Published by The Company of Biologists 2006
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Suppressors of cytokine signaling (SOCS) 1 and SOCS3 interact with and modulate fibroblast growth factor receptor signaling

Tal Ben-Zvi1,2, Avner Yayon3, Arieh Gertler2 and Efrat Monsonego-Ornan1,*

1 Institute of Animal Science, The Volcani Center, Bet Dagan 50250, Israel
2 Institute of Biochemistry, Nutritional Sciences, Faculty of Agricultural, Food and Environmental Quality, The Hebrew University of Jerusalem, Rehovot 76100, Israel
3 ProChon Biotech Ltd, PO Box 1482, Rehovot 76114, Israel



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  		Fig. 1. Expression of SOCS1 and SOCS3 mRNA in HEK-293T and RCS cells. The cells were stimulated with 50 ng/ml FGF and 5 µg/ml heparin for different periods of time (as indicated) after 8 hours in serum-free medium. Northern analysis was carried out on 2 µg mRNA hybridized with SOSC1 (socs1) or SOCS3 (socs3) probes.

 


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  		Fig. 2. hFGFR3 recruits SOCS1 and SOCS3 proteins. (A) HEK-293T cells transiently cotransfected with wild-type hFGFR3 (R3 WT), Myc-tagged SOCS1 (S1), or Myc-tagged SOCS3 (S3), as indicated. The amount of DNA was equalized with empty vector (EV). After 8 hours in serum-free medium, cells were treated with or without 50 ng/ml FGF and 5 µg/ml heparin for 5 minutes. (B) HEK-293 cells were transiently cotransfected with R3 WT or kinase-dead (KD) mutant and with EV, S1 or S3, as indicated. In both experiments, cells were dissolved in lysis buffer and total cell lysates were subjected to 7.5% SDS-PAGE followed by western blotting with anti-hFGFR3 antibody (Total lysates, IB: {alpha} hFGFR3). Cellular lysate protein (1 mg) was incubated overnight with anti-Myc agarose-conjugated antibody. Immunoprecipitated proteins were separated on 10% SDS-PAGE followed by western blotting with anti-Myc (IP: {alpha} cMyc, IB: {alpha} cMyc) or anti-hFGFR3 (IP: {alpha} cMyc, IB: {alpha} hFGFR3) antibodies. (C) HEK-293 cells were transiently transfected with R3 WT and five or ten times excess amount of KD mutant, as indicated. The DNA amount was equalized with empty vector. After 8 hours in serum-free medium, cells were stimulated with FGF9 for 5 minutes, lysed and subjected to IP with anti-hFGFR3 antibody. Immunoprecipitated proteins were separated on 10% SDS-PAGE followed by western blotting with anti-phosphotyrosine (IB: {alpha} P-Tyr) or anti hFGFR3 (IB: {alpha} hFGFR3) antibodies.

 


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  		Fig. 3. Expression patterns of the fluorescently tagged proteins. Confocal images (top) and confocal images merged with transmitted-light images (bottom). (I) HEK-293T cells transiently transfected with GFP-tagged hFGR3 (R3-GFP) together with Myc-tagged SOCS1 (S1-Myc) (C) or empty vector (A,B). (II) HEK-293T cells transiently transfected with CFP-tagged SOCS1 (S1-CFP) together with R3-GFP (F) or empty vector (D,E). After 8 hours in serum-free medium, cells treated with or without 50 ng/ml FGF and 5 µg/ml heparin for 1 hour. (III) HEK-293T cells transiently transfected with S1-CFP together with PRLR (G). After 8 hours in serum-free medium, cells were treated with ovine prolactin (PRL) for 1 hour. (IV) RCS WT and RCS S1 cells were pulsed with [35S]methionine for 30 minutes and treated with 20 ng/ml FGF and 5 µg/ml heparin. In the indicated periods of time, cells were lysed and subjected to immunoprecipitation with anti-hFGFR3 antibodies. Immunoprecipitated proteins were separated on 10% SDS-PAGE followed by fluorography. Bars, 10 µm.

 


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  		Fig. 4. SOCS1 inhibits STAT1 phosphorylation but elevates phosphorylated (p)MAPK levels in RCS cells. (A) RCS-WT and RCS-S1 cells were treated with 100 ng/ml FGF and 5 µg/ml heparin for different periods of time (as indicated) or with interferon-{alpha} (IFN{alpha}) after 8 hours in serum-free medium. Total cell lysates were subjected to 10% SDS-PAGE followed by western blotting with anti-pSTAT1 ({alpha} p-STAT1) and anti-STAT1 ({alpha} STAT1) antibodies. (B) RCS-WT and RCS-S1 cells were treated with 10 ng/ml FGF and 5 µg/ml heparin for different periods of time (as indicated) after 8 hours in serum-free medium. Total cell lysates were subjected to 10% SDS-PAGE followed by western blotting with anti-Myc ({alpha} c-Myc), anti-pMAPK ({alpha} pMAPK) and anti-MAPK ({alpha} MAPK) antibodies.

 


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  		Fig. 5. SOCS1 elevate OPN mRNA in RCS cells. (A) RCS WT and HEK-293T cells were stimulated with 50 ng/ml FGF and 5 µg/ml heparin for different periods of time (as indicated) after 8 hours of starvation with serum-free medium. Northern analysis of 2 µg mRNA hybridized with the OPN probe. (B) RCS WT cells and RCS cells stably transfected with Myc-tagged SOCS1 (RCS S1) were starved for 8 hours in serum-free medium and then treated as indicated with different concentration of FGF and heparin for 24 hours. Northern analysis of 10 µg total RNA hybridized with the OPN probe. The amount of RNA on the membrane was visualized by Methylene-Blue staining of the 18S ribosomal RNA.

 


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  		Fig. 6. PD decreases OPN level more dramatically in RCS SOCS1 than RCS WT cells. (A) RCS WT and RCS S1 cells were starved for 8 hours in serum-free medium, treated with the MAPKK inhibitor PD98059 (PD) or DMSO for 30 minutes and then treated with or without 20 ng/ml FGF and 5 µg/ml heparin for 15 minutes. Cell lysates were subjected to 10% SDS-PAGE followed by western blotting with anti-pMAPK ({alpha} pMAPK) and anti-MAPK ({alpha} MAPK) antibodies. (B) RCS WT and RCS S1 cells were starved for 8 hours in serum-free medium, treated with PD or DMSO for 30 minutes, and then treated as indicated with different concentration of FGF and heparin for 16 hours. Northern analysis of 10 µg total RNA hybridized with OPN probe. The amount of RNA on the membrane was visualized by Methylene-Blue staining of the 18S ribosomal RNA.

 

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