Vinculin mouse

For confocal microscopy of cell adhesions, EV1 and type I collagen were coated either on 6 cm cell culture plates inside paraffin circle, or on plastic coated glass surface. Cells were allowed to attach for 1 hour and then fixed in 4% paraformaldehyde for 20 minutes at room temperature, and permeabilized with 0.2% Triton x-100 in PBS for 5 minutes. Cells attached on cell culture plates were immunolabelled in 50 μl drop inside paraffin circle with α2 integrin (rat, clone 430903, R&D Systems), β1 integrin (clone K20, sc-18887, Santa-Cruz Biotechnology, Inc.), talin-1 (MAB1676, Chemicon), and vinculin (mouse, Sigma) antibodies where indicated. Alexa-488 or 555 labelled secondary antibodies against mouse and rat (Invitrogen), were used. Actin stress fibers were visualized with Alexa-633 conjugated phalloidin (Invitrogen). Mowiol, including DABCO (Sigma), was added on top of the cells and high quality 13 mm cover glass was placed inside a circle on a cell culture plate. For imaging, the plate was inverted, attached on objective glass from the bottom, and imaged with Zeiss LSM 510 confocal microscope. Shown confocal microscopy images are not adjusted.

For Western blotting and immunostainings, the following antibodies were used: VE‐CADHERIN rat (550548, BD Biosciences); VE‐CADHERIN goat (sc‐6458, Santa Cruz); PECAM1 hamster (MAB1398Z, Millipore); PECAM1 rat (553370, BD); PECAM1 rabbit (ab28364, Abcam); mKLF4 goat (AF3158, R&D); hKLF4 goat (AF3640, R&D); FSP1 rabbit (07‐2274, Millipore); ID1 rabbit (BCH‐1/37‐2, BIOCHECK); SCA1 rat (ab51317, Abcam); hCLAUDIN5 rabbit (ab53765, Abcam); pSMAD1/5 rabbit (9516, Cell Signaling); SMAD1 rabbit (9644, Cell Signaling); Glucose transporter type 1 (GLUT1) rabbit (RB9052P1, Thermo Scientific); ERK5 rabbit (07‐039, Upstate); VE‐PTP rabbit (produced and purified by New England Peptide); MEKK3 rabbit (5727, Cell Signaling); MEK5 mouse (610957, BD); BMP6 sheep (LS‐C150156, LS‐BIO); GAPDH mouse (SC‐32233, Santa Cruz); tubulin mouse (T9026, Sigma); vinculin mouse (V9264, Sigma); horseradish peroxidase (HRP)‐linked anti‐mouse, anti‐rat, and anti‐rabbit (Cell Signaling); and HRP‐linked anti‐goat (Promega). Biotin‐conjugated isolectin B4 (Vector Lab) was used to identify retinal vasculature. ALEXA FLUOR 488, 555, and 647 donkey secondary antibodies were from Life Technologies.