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Fig. 9. Expression of Q63L RhoB results in colocalization of CXCR2 with Rab4 and mannose-6-phosphate receptor (MPR). Confocal images of immunofluorescence-stained HEK293 cells stably expressing CXCR2. (A,B) CXCR2 and Rab4 staining in cells transfected with empty vector (A) or Myc-RhoB Q63L (B) and stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is Rab4, blue is Myc-RhoB Q63L. Bars, 10 µm. (C) Quantification of colocalization of CXCR2 with Rab4. Values shown are the mean ± s.e.m. ^*P<0.0005, significant differences (Student's t-test) of cells transfected with Myc-RhoB Q63L versus vector-transfected cells. (D,E) CXCR2 and MPR staining in cells transfected with empty vector (D) or Myc-RhoB Q63L (E) and stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 30 minutes. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is MPR, blue is Myc-RhoB Q63L. Bars, 10 µm. (F) Quantification of colocalization of CXCR2 with MPR. Values shown are the mean ± s.e.m. ^*P<0.0005, significant differences (Student's t-test) of cells transfected with Myc-RhoB Q63L versus vector-transfected cells. Quantification of the percentage of CXCR2 colocalized with Rab4 or MPR in 20 fields was performed using the MetaMorph Imaging system (Universal Imaging). Images were processed using Photoshop (Adobe Systems). Data shown are representative from three separate experiments.

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