Chicken anti mouse af488

HUVEC pools were purchased from Invitrogen (Breda, The Netherlands). EGM-2 medium and SingleQuots^™ for HUVEC cell culture were purchased from Lonza (Verviers, Belgium). PAI-1 inhibitors PAI-039 (tiplaxtinin) and TM5275 were purchased from Axon Medchem BV (Groningen, The Netherlands) and diluted in DMSO (referred to as solvent). PAI-1 rabbit polyclonal antibody was a kind gift from Dr. Sacha Zeerleder. VE-cadherin monoclonal mouse antibody, clone BV6, was purchased from Millipore (Amsterdam, The Netherlands) and actin [AC-40] monoclonal mouse antibody was purchased from Sigma (Zwijndrecht, The Netherlands). GM130 rabbit monoclonal antibody was from Cell Signaling (Leiden, The Netherlands). Goat polyclonal anti-VE-cadherin [C-19] and rabbit polyclonal anti-α-catenin were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Mouse monoclonal antibodies directed against mouse monoclonal PECAM-1 AF488 [WM59], VE-cadherin AF 647 [55-7H1], IgG1 AF674, β-catenin [14 (link)] and p120 catenin [98/pp120] were purchased from BD Biosciences (Amsterdam, The Netherlands). Texas Red-X Phalloidin, chicken anti-goat AF647, chicken anti-rabbit AF488, chicken anti-rabbit AF594, chicken anti-mouse AF488 and IgG1 AF488 were from Invitrogen (Breda, The Netherlands).

HeLa cells were grown to 80% confluency in 6-well plates and transfected with 300 ng pcDNA3.1-FLAG-RBM39 constructs using Lipofectamine2000 (Invitrogen). On the following day, 40,000 HeLa cells were re-seeded in 8-well chambers (Bioswisstec AG) and fixed after 24 h with 4% PFA for 30 min. After three washes with TBS, the cells were permeabilised and blocked using 1x TBS, 0.5% (v/v) Triton-X-100, 6% BSA at room temperature for 1 h. Ms anti-FLAG M2 antibodies (Sigma) were diluted 1:200 in TBS+/+ (1x TBS, 0.1% (v/v) Triton-X-100, 6% BSA) and incubated with the cells overnight at 4 °C. After 3 × 5 min washes with TBS+/+, the secondary antibody (Chicken anti-Mouse AF488, 1:500, Invitrogen) was diluted in TBS+/+ and bound to the primary antibody at 37 °C for 1.5 h followed by incubation at room temperature for 30 min. Then, the slides were washed 5 times with 1x TBS and mounted with Vectashield HardSet mounting medium containing DAPI (Vectorlabs). Images were acquired with a non-confocal fluorescence microscope (Leica, DMI6000 B) using the Leica Application Suite software (LAS-X) or with a non-confocal Eclipse Ti-2 epifluorescence microscope (Nikon) using the NIS-Elements AR software (Ver 5.01) using a 60x/1.4 NA oil immersion lens. For printing, brightness and contrast of the pictures were linearly enhanced.