Phospho stat3

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).