CXCR2 interacts with both PDZ1 and PDZ2 of NHERF1.

(A) GST pull-down of CXCR2 with NHERF1. Lysates of HEK293 cells overexpressing HA-tagged CXCR2 were used as prey. GST fusion proteins of NHERF1 PDZ1, PDZ2, and PDZ1-PDZ2 were used as bait. GST alone served as a negative control. Binding experiments were analyzed by SDS-PAGE and visualized by immunoblot using anti-HA antibodies. The amount of beads-immobilized GST proteins in each reaction is shown in the lower panel. (B) Biotin pull-down assays to detect direct interaction between CXCR2 and NHERF1. A biotinylated peptide corresponding to the last 13 residues of CXCR2 was used as bait, while purified GST-PDZ1, GST-PDZ2, GST-PDZ1-PDZ2 and GST alone as prey. Binding was resolved by SDS-PAGE and immunoblotted with anti-GST antibodies. (C) All experiments performed in (A) and (B) were repeated three times. The results were quantified using the CCD gel imager (UVP Chemidoc) and presented as mean±standard deviation. The asterisks indicate statistically significant differences (P < 0.05) between the values indicated by the brackets. Statistical analysis was performed using the two-tailed Student’s t-test. Top: GST pull-down of CXCR2 with NHERF1, and bottom: biotin pull-down of NHERF1 PDZ domains with the CXCR2 peptide.