Figure 4.
Figure 4. Ephrin B2 regulation by KS growth factors. (A) Inhibition of ephrin B2 in KS-SLK-vGPCR stable cell lines by neutralizing antibodies. Cells were cultured in full growth medium and exposed to antibodies as indicated in the figure (100 ng/mL) for 36 hours before collection and lysis for Western blot. Membranes were sequentially probed with antibody to ephrin B2, EphB4, and β-actin. Cell line 293 does not express ephrin B2 or EphB4 and serves as a negative control. Positive controls are 293 transiently transfected with either full-length ephrin B2 or EphB4 expression vectors (293/ephrin B2 and 293/EphB4, respectively). MAb indicates oncostatin M monoclonal antibody. (B) Immunoblots (IB) of protein extracts of KS-SLK cells were serum-starved for overnight and then treated with VEGF alone (25 ng/mL; lane 1), VEGF and VEGF antibody (50 ng/mL) premixed for 10 minutes and 20 minutes (lanes 2 and 3, respectively), or media alone (lane 4) for 20 minutes. (Top) Equal amounts of proteins from these samples were immunoprecipitated (IP) with VEGFR-2 antibody (50 ng/mL) on protein G sepharose and probed for antiphosphotyrosine monoclonal antibody (pTyr). The same blot was stripped and then probed for VEGFR-2. (Bottom) Lysates were also loaded directly and sequentially probed for p44/42 MAP kinase, phospho-p44/42 MAP kinase, and β-actin. (C) Cell viability assay in KS-SLK, T1 fibroblasts, and SCC-25 squamous cell carcinoma cells treated with neutralizing antibodies used in panel A. Note that VEGF-C antibody is not verified as neutralizing, instead VEGFR3/Fc chimera was used as a soluble decoy receptor to block VEGF-C binding to cells. Viability was measured by MTT after 48-hour exposure to antibodies or Fc fusion. Shown are means ± SEM of triplicate determinations. (D) Induction of ephrin B2 in HUVECs. Cells were cultured in EBM-2 (endothelial basal medium) supplemented with 5% FCS. This medium lacks the additional growth factors provided for EGM-2 (endothelial growth medium; see “Patients, materials, and methods” for details) in which HUVECs are routinely cultured. Individual growth factors were added as indicated and the cells harvested after 36 hours. Quantity of protein loading and transfer was determined by reprobing the membranes β-actin monoclonal antibody. NT indicates no treatment; and Onco-M, oncostatin-M.

Ephrin B2 regulation by KS growth factors. (A) Inhibition of ephrin B2 in KS-SLK-vGPCR stable cell lines by neutralizing antibodies. Cells were cultured in full growth medium and exposed to antibodies as indicated in the figure (100 ng/mL) for 36 hours before collection and lysis for Western blot. Membranes were sequentially probed with antibody to ephrin B2, EphB4, and β-actin. Cell line 293 does not express ephrin B2 or EphB4 and serves as a negative control. Positive controls are 293 transiently transfected with either full-length ephrin B2 or EphB4 expression vectors (293/ephrin B2 and 293/EphB4, respectively). MAb indicates oncostatin M monoclonal antibody. (B) Immunoblots (IB) of protein extracts of KS-SLK cells were serum-starved for overnight and then treated with VEGF alone (25 ng/mL; lane 1), VEGF and VEGF antibody (50 ng/mL) premixed for 10 minutes and 20 minutes (lanes 2 and 3, respectively), or media alone (lane 4) for 20 minutes. (Top) Equal amounts of proteins from these samples were immunoprecipitated (IP) with VEGFR-2 antibody (50 ng/mL) on protein G sepharose and probed for antiphosphotyrosine monoclonal antibody (pTyr). The same blot was stripped and then probed for VEGFR-2. (Bottom) Lysates were also loaded directly and sequentially probed for p44/42 MAP kinase, phospho-p44/42 MAP kinase, and β-actin. (C) Cell viability assay in KS-SLK, T1 fibroblasts, and SCC-25 squamous cell carcinoma cells treated with neutralizing antibodies used in panel A. Note that VEGF-C antibody is not verified as neutralizing, instead VEGFR3/Fc chimera was used as a soluble decoy receptor to block VEGF-C binding to cells. Viability was measured by MTT after 48-hour exposure to antibodies or Fc fusion. Shown are means ± SEM of triplicate determinations. (D) Induction of ephrin B2 in HUVECs. Cells were cultured in EBM-2 (endothelial basal medium) supplemented with 5% FCS. This medium lacks the additional growth factors provided for EGM-2 (endothelial growth medium; see “Patients, materials, and methods” for details) in which HUVECs are routinely cultured. Individual growth factors were added as indicated and the cells harvested after 36 hours. Quantity of protein loading and transfer was determined by reprobing the membranes β-actin monoclonal antibody. NT indicates no treatment; and Onco-M, oncostatin-M.

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