Colocalization of CXCR2 or LDL with Lamp-1 in HEK293 cells overexpressing Rab7 or Rab7-T22N.

(A-C) Colocalization of CXCR2 with LAMP-1. HEK293 cells stably cotransfected with plasmids encoding CXCR2 and vector (A), CXCR2 and Rab7 (B), or CXCR2 and Rab7-T22N (C) were treated with CXCL8 at 37°C for 4 hours. Cells were fixed with methanol and incubated with a mixture of a rabbit CXCR2 antibody and a mouse monoclonal Lamp-1 antibody for 30 minutes, followed by a mixture of an FITC-conjugated antirabbit antibody and a rhodamine-conjugated antimouse antibody for 30 minutes. Representative laser-scanning confocal micrographs from independent experiments demonstrating the distribution of CXCR2 (green) and Lamp-1 (red) and the colocalization of CXCR2 with Lamp-1 (yellow) are shown. (D-E) Colocalization of LDL with LAMP-1. HEK293 cells stably expressing Rab7 (D) or Rab7-T22N (E) were incubated with Dil-LDL (40 μg/mL) at 37°C for 4 hours, then fixed in methanol. Cells were incubated with a monoclonal LAMP-1 antibody followed by incubation with an FITC-conjugated antimouse antibody. Representative confocal micrographs from 3 independent experiments demonstrating the distribution of LDL (red), Lamp-1 (green), and the colocalization of LDL with Lamp-1 (yellow) are shown. Images were processed using Photoshop software. Bars, 10 μm.