Brij 97

The following reagents were used. Cys-specific chemical crosslinkers: BMH and TMEA (Thermo Scientific), o-PDM and p-PDM (Sigma) and Bis-MTS (methanethiosulfonate) reagents (M1M through M17M) (Toronto Research Chemicals). Detergents: Brij L23, Brij 97 and Brij 99 (Sigma), Triton-X-100, NP-40 and Tween-20 (MP Biochemical) and Digitonin (Sigma and Calbiochem). o-phenanthroline (Wako). Polyene antibiotics: nystatin (Wako) and natamycin (Fluka). Other chemicals were purchased from Sigma, Wako Pure Chemical, Nacalai Tesque and BD.

Cell surface biotinylation and immunoprecipitation procedures were performed as previously described [57 (link)]. Briefly, cells were incubated with PBS/2.5 mg EZ-LinkSulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA) at 4°C for 1 h and the biotinylation reaction was terminated by addition of Tris-HCl 50 mM, pH 7.5. Cells were lysed using 1% Brij97 (Sigma, St Louis, MO) buffer. After 30 min at 4°C, insoluble material was removed and the cell lysate was precleared for 2h using heat inactivated goat serum and protein G sepharose beads (GE Healthcare, England). The cell extracts were then incubated with control or specific mAb directly conjugated to sepharose 4B for 2 h, washed and eluted with Laemmli buffer. For re-immunoprecipitation experiments, associated proteins were eluted using a 1% Triton X100 buffer and then precipitated a second time with the indicated mAb-conjugated sepharose beads for 1 hour at 4°C.

To prepare whole cell extracts for immunoprecipitation, 2 × 10⁶ iBREC were detached by scraping, resuspended in 300 μl cold lysis buffer (40 mM TrisCl, 150 mM NaCl, 1% Brij 97 (Sigma-Aldrich), pH 7.4, supplemented with the EDTA-free protease inhibitor cocktail (Roche Diagnostics)), and incubated on ice for 1 hour. The supernatant resulting from subsequent centrifugation (15 min at 21100 × g, 4°C) was stored at -80°C [30 (link)]. These cell extracts were incubated with antibodies specific for CD9 or CD29 or an isotype-matched control IgG (final antibody concentrations: 10 μg/ml) under gentle agitation on ice for one hour. To precipitate formed protein-antibody complexes, Protein G Plus/Protein A-Agarose beads (15 μl per 1 μg antibody; Calbiochem/Merck, Darmstadt, Germany) were added. After incubation for one hour under constant agitation at 4°C, beads were separated by centrifugation (3 min for 200 × g) and washed three times with lysis buffer. Bound antibodies—free or attached to the targeted proteins—were removed from the beads by incubation with 1% sodium dodecyl sulfate (w/v) for 10 min at 60°C. Immunoprecipitates and whole cell extracts were analyzed by Western blot, and X-ray films (Hyperfilm ECL, GE Healthcare, VWR) were exposed to the resulting chemiluminescence (SuperSignal® Substrate kit; Thermo Fisher Scientific) for visualization of signals.