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Fig. 3. Expression of dominant-negative (T19N) RhoB alters trafficking of CXCR2 following 3 hours of CXCL8 stimulation. Confocal images of immunofluorescence staining of HEK293 cells stably expressing CXCR2. (A,B) Staining of CXCR2 and LAMP-1 in cells transfected with vector (A) or Myc-RhoB T19N (B) and stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 3 hours. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is LAMP-1, blue is Myc-RhoB T19N. Bars, 10 µm. (C) Quantification of colocalization of CXCR2 with LAMP-1 in cells transfected with vector, Myc-RhoB WT or Myc-RhoB T19N. Values shown are the mean ± s.e.m. ^*P<0.005, significant differences (Student's t-test) of vector or Myc-RhoB WT versus Myc-RhoB T19N transfected cells. (D,E) CXCR2 and Rab11a staining in cells transfected with empty vector (D) or Myc-RhoB T19N (E) stimulated with vehicle (Untreated) or 100 ng/ml CXCL8 for 3 hours. Transfected cells were identified by staining with anti-Myc antibody. Overlay images are pseudocolored; red is CXCR2, green is Rab11a, blue is Myc-RhoB T19N. Bars, 10 µm. (F) Quantification of colocalization of CXCR2 with Rab11a in cells transfected with vector, Myc-RhoB WT, or Myc-RhoB T19N. Values shown are the mean ± s.e.m. ^*P0.05, significant differences (Student's t-test) of vector or cells transfected with Myc-RhoB WT versus cells transfected with Myc-RhoB T19N. Quantification of the percentage of CXCR2 colocalized with LAMP-1 or Rab11a 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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