Figure 4

Analysis of expression of PARs on A549 cells by flow cytometry (A), Immunofluorescent microscopy (B) and RT-PCR (C). For PAR-1 and PAR-2 labeling, cells were incubated with PE-conjugated mouse anti-human PAR-1 monoclonal antibody and FITC-conjugated mouse anti-human PAR-2 monoclonal antibody for 30 min on ice. For PAR-3 and PAR-4 labeling, cells were incubated with rabbit anti-human PAR-3 or PAR-4 polyclonal antibodies for 30 min on ice followed by addition of FITC-conjugated goat anti-rabbit polyclonal antibodies. To detect cytoplasmic PARs, A549 cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature before analysis. Immunofluorescent microscopy was performed with a laser scanning confocal microscope. For RT-PCR analysis of expression of mRNAs of PARs in A549 cells, the gene products of PARs were separated in 1.5% agarose gels, stained with SYBR Green I Nucleic Acid Gel Stain and photographed under UV light. Lane1-6 represented DNA marker, PAR-1 (500 bp), PAR-2 (461 bp), PAR-3 (403 bp), PAR-4 (542 bp) and β-actin (148 bp), respectively.