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Fig. 6. Expression of GTPase-deficient RhoB (Q63L) mutant alters trafficking of CXCR2 following 30 minutes of CXCL8 stimulation. Confocal images of immunofluorescence-stained HEK293 cells stably expressing CXCR2. (A,B) CXCR2 and Rab11a staining in cells transfected with empty vector (A) or Myc-RhoB Q63L (B) 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 Rab11a, blue is Myc-RhoB Q63L. Bars, 10 µm. (C) Quantification of colocalization of CXCR2 with Rab11a in cells transfected with vector, Myc-RhoB WT or Myc-RhoB Q63L. Values shown are the mean ± s.e.m. ^*P<0.05, significant differences (Student's t-test) of vector or cells transfected with Myc-RhoB WT versus cells transfected with Myc-RhoB Q63L. (D,E) CXCR2 staining and EGFP-Rab7 in cells transfected with empty vector (D) and 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 EGFP-Rab7, blue is Myc-RhoB Q63L. Bars, 10 µm. (F) Quantification of colocalization of CXCR2 with EGFP-Rab7. Values shown are the mean ± s.e.m. ^*P<0.05, 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 Rab11a or EGFP-Rab7 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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