0.1%, blocked in 1% BSA for 30 min and stained with anti-pY397FAK (1:1000) overnight at 4 °C in 1% BSA. pFAK was visualized with goat-anti mouse-AF488 at 1:500 dilution (Invitrogen) and subsequently counterstained with phalloidin-TRITC (P-1951, Sigma). HGF surface staining was performed according to the manufacturer's instructions followed by IdoA staining (see above). Briefly, cells were fixed in methanol, and recombinant, biotinylated HGF (1:10) was added followed by visualization with avidin-FITC (1:10). Controls without biotinylated HGF or primary anti-IdoA antibodywere included in all experiments. Cells were analyzed using a Zeiss LSM 710 confocal scanning microscope equipped with a 20× and 63× objectives.
