Fig. 3. Association of Hip with CXCR2 or CXCR4 in intact cells.

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.