Localization of CXCR2 expression and of the expression of rab11⁺ endosomes by confocal analysis.

CXCR2-expressing HEK 293 cells were incubated with or without 1000 ng/mL GCP-2, at 37°C for 1, 5, and 60 minutes. The GCP-2–exposed cells were subdivided into 2 groups. One group of cells was washed and stained immediately after exposure to GCP-2 with anti-CXCR2 and anti-rab11–specific antibodies, as described in “Materials and methods.” The cells of the other group were washed and allowed to recover at 37°C for 90 minutes, in the presence or absence of 10 μg/mL cycloheximide. The cells were washed and stained with anti-CXCR2 and anti-rab11–specific antibodies, and subjected to confocal analysis as described in “Materials and methods.” In all the pictures shown, the red color represents the expression of CXCR2, as distinguished by the staining with rabbit antibodies against CXCR2, followed by rhodamine-conjugated antibodies against rabbit IgG. The green color, in all the pictures shown, represents the expression of rab11, as distinguished by the staining with mouse antibodies against rab11, followed by fluorescein isothiocipnate (FITC)-conjugated antibodies against mouse IgG. The yellow color indicates the colocalization of CXCR2 with rab 11 expression. (A) The expression of CXCR2 and rab11 prior to exposure to GCP-2. (B, C, D) The localization of CXCR2 and rab11 expression following the exposure of the cells to GCP-2 for 1, 5, and 60 minutes, respectively. (E) The expression of CXCR2 and rab11 after induction of internalization and recovery at 37°C for 1.5 hours in the absence of cycloheximide. (F) The same as in (E), in the presence of cycloheximide. Reference bars in the lower right corners of selected sections represent 10 μm. A representative experiment of 2 to 4 performed is shown.