FPR1 co-localizes with uPAR on the surface of uPAR-expressing HEK-293 cells.

<p>HEK-293 cells stably transfected with uPAR cDNA (uPAR-293 cells) were seeded on glass coverslips and transiently transfected with FPR1 cDNA. After 24 h, cells were incubated for further 24 h in culture medium (DMEM) containing 10% serum (Basal) or in DMEM without serum (−FBS). Prior to fixation, some serum-starved samples were stimulated for 1 h at 37°C, 5% CO2, with 10% FBS in DMEM (+FBS) or with 5 nM WKYMVm peptide (+W Pep). All samples were then fixed, incubated with the anti-uPAR monoclonal R4 antibody and the anti-FPR1 polyclonal antibody (<b>A</b>) or with the anti-uPAR monoclonal R4 antibody and the rabbit anti-β1 polyclonal antibody (<b>B</b>), stained with Cy3 or Alexa 488 conjugated secondary antibodies, and analyzed by confocal microscopy. The insets show a 4× magnification for the indicated region in each merge image. <b>C:</b> The degree of co-localization of the fluorescent signals was quantified on a minimum of 50 different cells by using the LSM 510 software. The number of co-localizing pixels was normalized to the total pixels of each fluorophore. Thus, the number of yellow pixels, corresponding to co-localizing uPAR-FPR1, has been normalized to green (uPAR) or red (FPR1) pixels shown in A and reported in 1^st and 2^nd set of columns, respectively. The number of pixels corresponding to co-localizing uPAR-β1 integrin has been normalized to green (uPAR) or red (β1 Integrin) pixels shown in B and reported in 3^rd and 4^th set of columns, respectively. (*) p≤0.05, as determined by the Student's <i>t</i> test.