Receptor internalization and recycling

HEK293 cells were harvested by accutase (Sigma-Aldrich) treatment and resuspended in PBS. Receptor internalization was induced by 15 min incubation with 20 nM soluble human CXCL16. To inhibit internalization, cells were pretreated for 15 min with 100 μM dynasore or 0.2% sodium azide (NaN₃), or the cells were incubated on ice during the whole procedure. For measurement of receptor recycling, cells were washed with pre-warmed PBS (37°C) and resuspended in PBS. Subsequently, cells were placed on ice, washed once with ice-cold PBS and fixed with 1% PFA. Receptor surface expression was investigated by antibody staining as described above. THP-1 cells were harvested and resuspended in PBS. Receptor internalization was induced by 15 min incubation with 1 nM CXCL16-Fc fusion protein at 37°C. As “untreated” control cells, cells were kept on ice during the preincubation as well as the complete recycling step. After internalization, cells were washed twice with PBS and kept at 37°C in PBS for the indicated time. Subsequently, cells were transferred on ice to stop the recycling process. Receptor expression of CXCR6 was investigated using CXCL16-Fc fusion protein as described above.