Fluorescein isothiocyanate conjugated transferrin

HUVECs were cultured in plastic dishes that were obtained from ibidi, 35 mm round, appropriate for microscopy, previously coated with collagen type I. For internalization experiments, cells were washed 3 times with blocking buffer, treated for 30 min with vehicle or inhibitors in blocking buffer, transferred to 37 °C and incubated with 50 μg/mL fluorescein isothiocyanate-conjugated transferrin (Invitrogen). Then, the samples were washed 3 times with Ca²⁺/Mg²⁺ HBSS, fixed in 3.7% PFA for 20 min and analyzed by microscopy. Images of the cells were captured using a Leica TCS SP5 II scanning confocal microscope and a Leica 63X HCX PL APO 1.4 NA objective. Imaging data were subsequently processed using the LAS AF software, according to the manufacturer’s guidelines.

Recombinant human VEGFΑ₁₆₅ was obtained from Immunotools. Mouse anti-VEGFR2 extracellular domain monoclonal antibodies were from Abcam, whereas the rabbit monoclonal and polyclonal antibodies were from Cell Signalling and Santa Cruz Biotechnology, respectively. Fluorescein isothiocyanate-conjugated transferrin was from Invitrogen. The anti-actin antibody was from Millipore, whereas the anti-clathrin heavy chain antibody was from BD Biosciences. The rabbit polyclonal antibody against Early Endosome Antigen 1 (EEA1) was kindly provided by Marino Zerial (MPI-CBG, Dresden, Germany). The antibodies against ERK1/2, p-ERK1/2, PLCγ, p-PLCγ and p-VEGFR2 (1175) were from Cell Signalling whereas the antibody against p-VEGFR2 (1214) was from Invitrogen. Secondary antibodies coupled to Alexa fluorophores were from Invitrogen, while the HRP conjugated antibodies were from Jackson Immunoresearch. All other reagents were obtained from Sigma-Aldrich, unless stated otherwise.