Quantitative Western Blotting of Cell Signaling Proteins

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).