Figure 1
Figure 1. rDrm induces Ang-1 expression in EC in a NF-κB–dependent manner. (A) Serum-starved SIE cells were stimulated for 2, 6, 12, and 24 hours with 50 ng/mL of rDrm. At the end of incubation, total RNA was extracted and Ang-1 and Ang-2 transcripts were analyzed by semiquantitative RT-PCR. β-Actin was used as a control. (B) Cell lysates were prepared from SIE cells stimulated for 0, 6, and 18 hours with 50 ng/mL of rDrm. Then, 20-μg aliquots were probed by Western blotting with anti–Ang-1 antibodies (Santa Cruz Biotechnology). Uniform loading of the gels was confirmed by incubation of the membranes with anti–focal adhesion kinase (FAK) antibodies. (C) Serum-starved ECs were incubated for 20 minutes with 50 ng/mL of rDrm and immunostained with an anti-RelA/p65 antibody (Santa Cruz Biotechnology) followed by antirabbit FITC (Invitrogen). Control cells show a diffuse cytoplasmic RelA/p65 immunoreactivity (top panel) that localizes into the nucleus after rDrm stimulation (bottom panel). Analysis was performed using a Zeiss Axiovert 200M epifluorescence microscope equipped with a Plan-Apochromat 63×/1.4 NA oil objective. (D) SIE cells were stimulated for 0 to 30 minutes with 50 ng/mL of rDrm. Then, nuclear (5 μg) and cytoplasmic (10 μg) extracts were probed by Western blotting with anti-RelA/p65 and anti-IkBα (Santa Cruz Biotechnology) antibodies, respectively. Uniform loading of the gels was confirmed by incubation of the membranes with antihistone H3 (Santa Cruz Biotechnology) and anti-FAK antibodies, respectively. (E) Nuclear extracts (2 μg) of SIE cells treated for 0 to 30 minutes with 50 ng/mL of rDrm were incubated with a biotin-labeled NF-κB double-stranded DNA oligonucleotide probe. (Left panel) The protein-DNA complexes were analyzed by EMSA onto a native 6% polyacrylamide gel; the first lane shows the migration of the labeled probe in the absence of nuclear extract. (Right panel) Densitometric analysis of the protein-DNA complexes shown in the left panel; data are the mean plus or minus SEM of 3 independent experiments. (F) SIE cells were treated for 1 hour with NF-κB inhibitors SN50 (45 pg/mL, US Biological, Swampscott, MA) or BAY 11-7085 (5 μM; Sigma-Aldrich, St Louis, MO) before rDrm administration. After 18 hours, cell extracts were probed by Western blotting with anti-Ang-1 antibodies (a). In addition, conditioned media from the corresponding cell cultures were added for 10 minutes to naive serum-starved SIE cells. Then, immunoprecipitation with anti–Tie-2 antibodies was performed on the cell extracts followed by Western blotting with antiphosphotyrosine antibodies (Santa Cruz Biotechnology) (b). Uniform loading of the gel was confirmed by incubation of the membrane with anti–Tie-2 antibodies. Similar results were obtained in 2 independent experiments.

rDrm induces Ang-1 expression in EC in a NF-κB–dependent manner. (A) Serum-starved SIE cells were stimulated for 2, 6, 12, and 24 hours with 50 ng/mL of rDrm. At the end of incubation, total RNA was extracted and Ang-1 and Ang-2 transcripts were analyzed by semiquantitative RT-PCR. β-Actin was used as a control. (B) Cell lysates were prepared from SIE cells stimulated for 0, 6, and 18 hours with 50 ng/mL of rDrm. Then, 20-μg aliquots were probed by Western blotting with anti–Ang-1 antibodies (Santa Cruz Biotechnology). Uniform loading of the gels was confirmed by incubation of the membranes with anti–focal adhesion kinase (FAK) antibodies. (C) Serum-starved ECs were incubated for 20 minutes with 50 ng/mL of rDrm and immunostained with an anti-RelA/p65 antibody (Santa Cruz Biotechnology) followed by antirabbit FITC (Invitrogen). Control cells show a diffuse cytoplasmic RelA/p65 immunoreactivity (top panel) that localizes into the nucleus after rDrm stimulation (bottom panel). Analysis was performed using a Zeiss Axiovert 200M epifluorescence microscope equipped with a Plan-Apochromat 63×/1.4 NA oil objective. (D) SIE cells were stimulated for 0 to 30 minutes with 50 ng/mL of rDrm. Then, nuclear (5 μg) and cytoplasmic (10 μg) extracts were probed by Western blotting with anti-RelA/p65 and anti-IkBα (Santa Cruz Biotechnology) antibodies, respectively. Uniform loading of the gels was confirmed by incubation of the membranes with antihistone H3 (Santa Cruz Biotechnology) and anti-FAK antibodies, respectively. (E) Nuclear extracts (2 μg) of SIE cells treated for 0 to 30 minutes with 50 ng/mL of rDrm were incubated with a biotin-labeled NF-κB double-stranded DNA oligonucleotide probe. (Left panel) The protein-DNA complexes were analyzed by EMSA onto a native 6% polyacrylamide gel; the first lane shows the migration of the labeled probe in the absence of nuclear extract. (Right panel) Densitometric analysis of the protein-DNA complexes shown in the left panel; data are the mean plus or minus SEM of 3 independent experiments. (F) SIE cells were treated for 1 hour with NF-κB inhibitors SN50 (45 pg/mL, US Biological, Swampscott, MA) or BAY 11-7085 (5 μM; Sigma-Aldrich, St Louis, MO) before rDrm administration. After 18 hours, cell extracts were probed by Western blotting with anti-Ang-1 antibodies (a). In addition, conditioned media from the corresponding cell cultures were added for 10 minutes to naive serum-starved SIE cells. Then, immunoprecipitation with anti–Tie-2 antibodies was performed on the cell extracts followed by Western blotting with antiphosphotyrosine antibodies (Santa Cruz Biotechnology) (b). Uniform loading of the gel was confirmed by incubation of the membrane with anti–Tie-2 antibodies. Similar results were obtained in 2 independent experiments.

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