CCR5 Immunoprecipitation Assay Protocol

The immunoprecipitation assay was carried out as previously described [10 (link),11 (link),14 (link)]. Briefly, the cells were treated with CCR5 Ab Pos and relative controls, then were collected, washed twice in PBS and lysed with cold lysis buffer (20 mM Tris-HCl, pH 8, 1 mM EDTA, 200 mM NaCl, 1% Nonidet P-40, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM NaF, 0.1 μg/mL Protease Inhibitors). The protein samples were precleared with 50% of protein-A slurry for 16 h. Immunoprecipitations were performed with the indicated antibodies pre-adsorbed on protein A-Sepharose beads (Amersham Pharmacia Biotech AB) for 2 h at 4 °C [10 (link),11 (link),14 (link)]. In particular, the monoclonal antibody anti-CKR-5 (D6) recognizes ECL2 and ECL3 of CCR5, thus it does not interfere with the activity of CCR5 Ab Pos, which are specific for ECL1 domain of CCR5 only (unpublished data). After overnight incubation with the protein extracts, complexate-beads were resolved by SDS–PAGE and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Immunoblotting was performed with β-arrestin1/2, ERK1 and Rab5 antibodies and immunoreactivity was revealed by using secondary antibodies specific for IP (Abcam). The chemiluminescent signals were detected by ECL method (Millipore).