Protein a sepharose beads

BMM cells were incubated with 500 ng/mL of mRANKL-WT or mRANKL-MT3 at 37 °C for 45 min, and then cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.6–8.0, 100 mM sodium chloride NaCl, 300 mM sucrose, 3 mM MgCl₂ [buffer A]; and 20 mM Tris pH 8.0, 100 mM NaCl, 2 mM ethylenediaminetetraacetic acid [buffer B]). Whole cell lysates obtained by centrifugation were incubated with antibodies specific for RANK (Cell Signaling Technology, Danvers, MA, USA, #14373S) and LGR4 (MyBioSource, San Diego, CA, USA, #MBS468030) (dilution 1:100) and protein A Sepharose beads (Amersham Biosciences, Little Chalfont, UK) for 2 h at room temperature. The immune complexes were washed three times using wash buffer and examined by western blotting.

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of CCR5, thus it does not interfere with the activity of CCR5 Ab Pos, which are specific for ECL1 domain of CCR5 only (unpublished data).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).

For ERα-immunoprecipitation, HeLa cells (ATTC) were transfected with 3 μg hERα-pSG5 or pSG5 using Lipofectamine^® 2000 (Thermo Scientific) and OptiMEM (Invitrogen). 24 h after transfection, cells were stimulated for 24 h with E2 and/or C18:0, in concentrations as described for THP-1 cells. Cells were lysed with RIPA (with proteinase-inhibitors, Complete Mini, Roche), sonicated (Sonopuls HD 2070, 30 s, 40–50%), and centrifuged. Supernatant was used to perform immunoprecipitation (IP) with anti-ERα-antibody (D8H8, Cell Signaling) bound to Protein A Sepharose-beads (Amersham). For western immunoblotting a distinct anti-ERα-antibody (sc-8002, Santa Cruz) was used. Washed beads-antibody-ERα complexes were analysed for fatty acids- binding as described above.

ChIP assays were performed as per the manufacturer’s protocol (Upstate Biotechnology). Briefly, 10 × 10⁶ cells were cross-linked with formaldehyde (1%), sonicated at 4 °C (5 pulses for 10 seconds each at 30% amplitude) and centrifuged at 13,000 rpm at 4 °C for 10 minutes to obtain clear lysate. Cell lysates were pre-cleared for 2 h with protein-A–Sepharose beads (Amersham Biosciences) and incubated overnight with indicated antibodies. The immune complexes were pulled down using protein-A–Sepharose beads pre-blocked with BSA and salmon sperm DNA. After multiple washing steps in low salt, high salt and lithium chloride containing buffers, DNA was eluted using elution buffer made in 0.1 M NaHCO₃ and 1% SDS followed by purification using phenol-chloroform extraction. The eluted DNA was analyzed using real time quantitative PCR (qPCR) as described above and specific primers.