Specific staining of CXCR2 and colocalization of CXCR2 with endogenous Rab5, Rab11a, and Rab7 in HEK293 cells.

For the specific staining of CXCR2, cells stably expressing (A) or not expressing (B) CXCR2 were fixed in methanol. Cells were incubated with a monoclonal CXCR2 antibody for 30 minutes, followed by incubation with an FITC-conjugated antimouse antibody for 30 minutes. Specific cell surface expression of CXCR2 was determined by confocal microscopy. For the colocalization of CXCR2 with the endogenous Rab proteins, cells stably expressing CXCR2 were treated with CXCL8 (200 nM) for 1 hour (C-F) or 4 hours (G-H), then fixed in methanol. Cells were incubated with a mixture of a monoclonal CXCR2 antibody and a rabbit antibody for Rab5 (C-D), Rab11a (E-F), or Rab7 (G-H), for 30 minutes. After washing with PBS, cells were incubated with a mixture of an FITC-conjugated antimouse antibody and a rhodamine-conjugated antirabbit antibody for 30 minutes. Representative confocal micrographs from 3 independent experiments demonstrating the intracellular localization of CXCR2 (C,E,G), Rab5 (D), Rab11a (F), and Rab7 (H) are shown. Arrows indicate the colocalization of CXCR2 with the individual Rab proteins. Bars, 10 μm.