Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Figure S1 - Time course and dose dependency of VEGF effects on ERK2/1 phosphorylation in oFPAE cells. Serum-starved subconfluent oFPAE cells were treated with 10 ng/ml VEGF at indicated times (A) or with increasing concentrations of VEGF at 5 min (B). Total or phosphorylated ERK2/1 was analyzed with specific antibodies.
• Supplemental Figure S2 - Role of Ca²⁺, PKC, and Raf-1 in ERK2/1 phosphorylation by VEGF in oFPAE cells. A & B, Ca²⁺ in VEGF (A) or PMA (B) induced ERK2/1 activation. oFPAE cells were treated with BAPTA/AM at indicated concentrations for 1 hr prior to VEGF (10 ng/ml) (A) or PMA (20 nM) (B) treatment for 5 mins. C, PKC in VEGF or PMA induced ERK2/1 activation. oFPAE cells were treated with PKC inhibitor, GF109203X at indicated concentrations for 1 hr prior to VEGF (10 ng/ml) or PMA (20 nM) treatment for 5 mins. D, Raf-1 in VEGF-induced ERK2/1 activation. oFPAE cells were treated with Raf-1 inhibitor, GW5074 at indicated concentrations for 1 hr prior to VEGF (10 ng/ml) treatment for 5 mins. E, VEGF stimulates PKCα/β_II and Raf-1 phosphorylation. oFPAE cells were treated with or without VEGF (10 ng/ml) for 5 mins. Cell extracts were prepared and subjected to western-blot analysis using specific antibodies. Bars represent mean ± SD of 4 separate experiments.
• Supplemental Figure S3 - Effect of angiogenic factors VEGF and FGF2 on caveolin-1 mRNA and protein expression in oFPAE cells. A. VEGF on caveolin-1 mRNA expression. oFPAE cells were treated with 10ng/ml VEGF for 24 hrs, total RNA were reverse transcribed and used for measuring caveolin-1 mRNA levels by PCR as described in Methods. L-19 mRNA was measured as a loading control. 1 & 2, no-RT controls. B, VEGF and FGF2 on caveolin-1 protein expression. oFPAE cells were treated with 10 ng/ml of VEGF or FGF2 (R&D System) for 24, 48 or 72 hrs. Total protein extracts were prepared for measuring caveolin-1 protein by Western blotting with specific antibodies. β-actin protein was measured as a loading control. Typical images representative of more than ten different experiments with similar results are shown for both panels. No difference was found in caveolin-1 expression by VEGF and FGF2.
• Supplemental Figure S4 - VEGFR2 interacts with caveolin-1 in caveolae of COS-7 cells. COS-7 cells were transfected with phKDR/CE4.1 or vector plasmid for 48 hrs prior to sucrose gradient centrifugation. Fractions (1 ml each) were analyzed by Western blotting with specific antibodies as indicated.