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Blood, Vol. 111, Issue 5, 2674-2680, March 1, 2008
Semaphorin 3A suppresses VEGF-mediated angiogenesis yet acts as a vascular permeability factor
Blood Acevedo et al.
111: 2674
Supplemental materials for: Acevedo et al
Supplemental materials and methods In vitro permeability studies
In vitro permeability studies were performed in as described previously.1 Fluorescence was quantitated using a Tecan Genios Pro and Magellan Software (Tecan, Swizterland). VE-cadherin staining
HUVECs were starved in serum-free MCDB 131 media for 1 hour prior to treatment with Sema3A (500ng/ml) or VEGF165 (50ng/ml). Cells were fixed in 2% paraformaldehyde, permeabilized and stained with anti-VE-cadherin (C-19, 2µg/ml), followed by 488-anti-goat secondary (Invitrogen). Images were acquired using laser scanning confocal microscopy with 60×/1.4 NA oil objective (Nikon C1si, Nikon Instruments Inc). Akt1/2 phosphorylation
Confluent BAECs (p4-6; Lonza) were starved in serum-free DMEM media (Lonza) for 16 hours prior to 10 min stimulation with Sema3A or VEGF165. Cells were lysed in RIPA buffer and immunoblotted with anti-P-Akt1/2 (PS473, Cell Signaling) and anti-Akt1/2 (BD Biosciences). Film was scanned on an Epson 1680 scanner (Epson, Long Beach, CA) using Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). REFERENCES 1. Chandra A, Barillas S, Suliman A, Angle N. A novel fluorescence-based cellular permeability assay. Journal of Biochemical and Biophysical Methods. 2007;70:329-333.
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