HEK293 cells stably expressing CXCR2 were transiently transfected with pcDNA3.1, His-tagged wild-type Hip, or His-tagged ΔTPR. Two days after transfection, cells were compared for chemotaxis in response to CXCL1 stimulation as described under “Experimental Procedures.” Values represent the mean ± S.E. of three different experiments. The data were analyzed using Student’s paired t test (*, p < 0.05).

A, diagrams of wild-type and mutant Hip forms, highlighting structurally distinct regions. WT, wild-type. B, the wild-type and mutant Hip forms in pcDNA3.1 were transiently transfected in HEK293 cells. Cells were lysed, and proteins were separated by 10% SDS-PAGE. The expression of His-tagged Hip forms was determined by Western blotting using a specific anti-His antibody. C, the GST-CXCR2 C terminus fusion proteins were incubated with the cell lysate of HEK293 cells transiently expressing His-tagged Hip forms. Co-precipitated Hip forms were analyzed by Western blotting using anti-His antibody as described in the Fig. 1B legend.

HEK293 cells stably expressing CXCR2 and transiently expressing His-tagged Hip were treated with carrier buffer (A–C) or CXCL8 (B–D) for 5 min. The cells were then fixed with methanol, incubated with a mixture of a mouse monoclonal anti-CXCR2 antibody and a rabbit polyclonal anti-His antibody for 30 min, then incubated with a mixture of a FITC-conjugated anti-mouse antibody and a rhodamine-conjugated anti-rabbit antibody for 30 min. Representative laser-scanning confocal micrographs demonstrating the distribution of CXCR2 (green) (A and D), His-tagged Hip (red) (B and E), and overlay (yellow) (C and F) are shown. Images were processed using Photoshop software.

HEK293 cells stably expressing CXCR2 were transiently transfected with vector (lanes 1 and 2), His-tagged wild-type Hip constructs (lanes 3 and 4), or His-tagged ΔTPR constructs (lanes 5 and 6). Cells were treated with carrier buffer (lanes 1, 3, and 5) or CXCL8 (100 ng/ml) (lanes 2, 4, and 6) for 10 min. 30 μl of protein from clarified cell lysate was loaded on SDS-polyacrylamide gel per lane and electrophoresed, and Western blot analysis was performed with a phosphospecific MAPK antibody. The blots were striped and re-probed with an antibody against ERK-2 and with an antibody against His, respectively, to ensure equal loading and protein expression.

A, yeast strain Y190 co-transformed with PACT2/Hip and PAS2/CXCR2-(311–355) or PAS2/CXCR2-(331–355) were replica-plated on SD medium without tryptophan, leucine, or histidine. The co-transformation of p53 and SV40 provided by the manufacturer was performed as a positive control (left panel). A β-galactosidase assay was carried out using a YPD plate replicate (right panel) as described under “Experimental Procedures.” B, the indicated GST or GST-CXCR2 C terminus fusion proteins were incubated with the cell lysate of HEK293 cells then absorbed onto glutathione-Sepharose beads. After washing, the beads were resuspended in loading buffer. Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Co-precipitated Hip proteins were analyzed by immunoblotting with a polyclonal Hip antibody. The membrane was stripped and reblotted with a mouse monoclonal GST antibody to confirm the protein expression and equal loading.

A, HEK293 cells stably expressing CXCR2 were transiently transfected with pcDNA3.1, His-tagged wild-type Hip, or His-tagged ΔTPR. Cells were incubated with 75 nCi/ml ¹²⁵I-CXCL8 at 4 °C for 1 h. Unbound ¹²⁵I-CXCL8 was removed by washing at 4 °C. The cells were warmed to 37 °C for the indicated time period. ¹²⁵I-CXCL8 remaining at the cell surface was removed with acetic acid (0.2 M, pH. 2.5) containing 0.5 M NaCl, and the internalized ¹²⁵I-CXCL8 was quantitated on a γ-counter. Values represent the mean ± S.E. of three independent experiments performed in duplicate. The data were analyzed using Student’s paired t test (*, p < 0.05). B, RBL-2H3 cells were transiently transfected with pcDNA3.1, His-tagged wild-type Hip, or His-tagged ΔTPR. Cells were treated with CXCL12 at 37 °C for 30 min, then were incubated with a monoclonal c-Myc antibody at 4 °C for 1 h. Cells were washed with ice-cold medium followed by incubation with fluorescein isothiocyanate-labeled goat anti-mouse IgG at 4 °C for 30 min. Cells were washed and fixed in 2% formaldehyde in phosphate-buffered saline and analyzed in FACScan. The staining of cells in the absence of primary c-Myc antibody (thin broken line) or with c-Myc antibody in cells incubated with (solid line) and without (thick broken line) CXCL12 are shown.

A, RBL-2H3 cells stably expressing CXCR2 were exposed to CXCL8 (100 ng/ml) for the indicated times, and CXCR2 was immunoprecipitated from the cell lysate using a rabbit polyclonal anti-CXCR2 antibody. A preimmune rabbit serum (Mock) was used in a parallel experiment to confirm the specificity of the immunoprecipitation. Proteins were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Co-precipitated Hip proteins were detected using a polyclonal Hip antibody. The membrane was stripped and reblotted with a mouse monoclonal anti-CXCR2 antibody to confirm equal loading. The results represent one of three independent experiments. B, human neutrophils prepared from a single donor were treated without (lane 1) or with (lane 2) CXCL8 (100 ng/ml) for 5 min. The cells were lysed, and CXCR2 was immunoprecipitated described above. Co-precipitated Hip was blotted using specific anti-Hip antibody. The membrane was stripped and reblotted with a mouse monoclonal anti-CXCR2 antibody (E2, Santa Cruz Biotechnologies) to confirm equal loading. C, HEK293 cells stably expressing wild-type (lanes 1 and 2) or I323A/L324A mutant form of CXCR2 (lanes 3 and 4) were treated with carrier buffer (lanes 1 and 3) or CXCL8 (100 ng/ml) (lanes 2 and 4) for 5 min. CXCR2 was immunoprecipitated and co-precipitated Hip proteins were analyzed as described in the Fig. 3A legend. D, HEK293 cells transiently transfected with myc-tagged CXCR4 were treated with carrier buffer (lane 1) or CXCL12 (100 nM) (lane 2) for 5 min. CXCR4 was immunoprecipitated using a monoclonal anti-myc antibody, and co-precipitated Hip proteins were analyzed as described in the Fig. 3A legend. IP, immunoprecipitation; IB, immunoblotting.