IQGAP1 is a novel CXCR2 interacting protein.

(A) IQGAP1 co-immunoprecipitates with CXCR2. Lysates from differentiated HL-60 cells expressing CXCR2 stimulated with vehicle (Mock, Untreated) or cells stimulated with 100 ng/ml CXCL8 for 1 min or 5 min were incubated with either normal rabbit IgG- (Mock IgG) or rabbit anti-CXCR2 antibody-coupled sepharose. Beads were washed and immunoprecipitated proteins were eluted with Laemmli sample buffer. Samples were analyzed by SDS-PAGE and western blot (IB) for CXCR2 and IQGAP1. (B) CXCR2 co-immunoprecipitates with IQGAP1-reverse co-immunoprecipitation. Cell lysates were prepared as described above and incubated with either normal rabbit IgG (Mock IgG) or polyclonal rabbit anti-IQGAP1 antibody. Immunoprecipitated proteins were analyzed by SDS-PAGE and western blot for IQGAP1 and CXCR2. (C & D) Co-localization of CXCR2 and IQGAP1. Immunofluorescence confocal images of CXCR2 and IQGAP1 staining in differentiated HL60 cells stably expressing CXCR2 and stimulated with vehicle (0 min) or 100 ng/ml of CXCL8 for 1 min, 5 min, and 30 min. Cells were stained with rabbit polyclonal anti-CXCR2 and mouse monoclonal anti-IQGAP1 antibodies, and incubated with species specific Cy2- and Cy3-conjugated secondary antibodies. Co-localization is seen at each time point. Overlay images are pseudo-colored where green is IQGAP1 and red is CXCR2. Image represents a single Z-section of 0.28 µm. Insets are enlarged 2X from original images. (E) Co-localization of IQGAP1 and CXCR2 in human neutrophils. Purified human neutrophils were induced to polarize in a Zigmond chamber under a gradient of CXCL8 (the arrows indicate the direction of gradient) for 20 min at 37°C. Panels a-c show three representative confocal immunofluorescence images of cells where cells are stained with antibodies against IQGAP1 (red) and CXCR2 (green). Panel d shows images of cells stained with normal IgG (isotype control). Scale bar  = 10 µm. Quantitation of percent co-localization of IQGAP1 and CXCR2 was plotted in panel F.