Figure 1
Expression of REEP5 and REEP6 enhances IL-8 receptor CXCR1-mediated signaling.
(A) HEK293/Gα15 cells transfected with the SRE-luc reporter, CXCR1 plasmids, and REEP were treated with 10 nM IL-8 and assayed for luciferase activity. **p < 0.01. NT; no treatment, V; vector, R; REEP. (B) Luciferase activity was assayed using cells expressing CXCR2. (C) Total RNA was extracted from HEK293 cells and expression of REEP was determined by RT-PCR. The expected PCR product sizes are indicated by an asterisk. (D) HEK293 cells transfected with SRE-luc, CXCR1, and either REEP5 or REEP6 were treated with 10-fold serially diluted IL-8 (from 602 nM) and assayed for luciferase activity. *p < 0.05, **p < 0.01. (E) Lysates from HEK293 cells transfected with HA-CXCR1 and REEP5 or REEP6, or HA-CXCR2 and REEP5 or REEP6 were applied for immunoprecipitation with anti-HA agarose and analyzed by western blotting with relevant antibodies. (F) Schematic representation of CXCR1 and CXCR2. Shaded regions represent the transmembrane domains. The chimeric receptors were generated by PCR. The designated numbers are the chimeric position. Dark gray indicates the transmembrane domain. (G) A co-immunoprecipitation assay was performed with the HA-tagged chimeric CXCR1/2 receptors and REEP6, and analyzed by western blotting with anti-REEP6 antibodies. HA-R, HA-CXCR. All experiments were performed at least three times.
