CXCR7 Scavenges CXCL12 Figure 2. Ectopic expression of CXCR7 and CXCR4 on MDCK cells. (A) MDCK were stably transfected with empty vector (Mock), with CXCR7, CXCR4, a vector coding for a CXCR7 lacking the cytoplasmic C-terminus (DCXCR7), and a vector coding for a chimeric CXCR7 containing the DRYLAIV motive of CXCR4. Receptor expression was determined by FACS analysis using saturating antibody concentrations (see Methods). (B) Confocal immunofluorescence analysis of unfixed MDCK cells expressing CXCR7 (upper panels) or CXCR4 (lower panels). Cells also expressed N-ter-Lck mCherry as membrane marker (red fluorescence). Left panels: confocal images of planes cut through intracellular regions of MDCK monolayers. Right panels: x-z planes reconstructed from confocal x-y stacks. For receptor (green) detection anti-CXCR7 (11G8 R&D) or anti CXCR4 (MAB173 R&D) were used. Receptor-bound primary antibodies were revealed with goat anti mouse IgG conjugated with Alexa488 (green fluorescence). doi:10.1371/journal.pone.0009175.g002 From the above experiments it is not clear if cycling of CXCR7 from the plasma membrane to endosomes is ligand induced or may also occur constitutively. To address this question we measured the dynamics of CXCR4 and CXCR7 expression at the plasma membrane in the absence of chemokines. Daudi cells or MDCK cells stably transfected with CXCR7 or CXCR4 were treated on ice with proteinase K. The protease is active at the low temperature, although with delayed kinetics, but membrane trafficking is completely abrogated [36]. Proteinase K cleaves the N-termini which contain the epitopes of CXCR7 for the mAb 9C4 PLoS ONE | www.plosone.org 5 February 2010 | Volume 5 | Issue 2 | e9175