Odyssey imaging studio software

Western Blotting was performed as described before^{46 (link)}. After overnight incubation with primary antibodies: Cx43 (Cell Signaling Technology, Danvers, MA, USA, 1:1000); GAPDH (abcam, Cambridge, UK, 1:5000); VE-Cadherin (Cell Signaling Technology, Danvers, MA, USA, 1:1000); Alpha-Tubulin (Sigma-Aldrich, Zwijndrecht, The Netherlands, 1:10000); Cav-1 (abcam, Cambridge, UK, 1:10000), membranes were washed with phosphate buffered saline containing tween (PBS-T) and incubated with the appropriate secondary antibody (IRdye, Licor, Bad Homburg, Germany) for 1 hour at room temperature. After a final wash, membranes were scanned with the odyssey infrared imaging system (Licor, Bad Homburg, Germany) and quantification of the signals was performed with Odyssey Imaging Studio software (Licor, Bad Homburg, Germany).

Western blotting was performed as described previously [19 (link), 20 (link)]. In order to obtain all membrane-bound proteins in the cell lysate, we performed ultrasonic lysis by sonification with each sample. Overnight incubation with primary antibodies was performed for caveolin-1 (Abcam, Cambridge, UK 1:20,000); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, UK, 1:5000); HIF1α (Acris, Novus Biological, UK, 1:1000); phosphoERK1/2 (Cell Signalling, Danvers, USA, 1:5000), ERK1/2 (Cell Signalling, Danvers, USA, 1:5000), phospho-eNOS and eNOS (Cell Signalling, Danvers, USA, 1:1000), phosphoSTAT3 (R&D Systems, Minneapolis, USA, 1:1000) or against STAT3 (R&D Systems, Minneapolis, USA, 1:1000).
Membranes were washed for 3 × 5 min in tris-buffered saline containing Tween buffer (TBST) before incubating with the appropriate secondary antibody (IRdye, LI-COR, Bad Homburg, Germany) for 1 h at room temperature. After a final washing, membranes were scanned with the Odyssey infrared imaging system (LI-COR, Bad Homburg, Germany), and quantification of the signals was performed with Odyssey Imaging Studio software (LI-COR, Bad Homburg, Germany).