Biotinylation and immunoprecipitation

The surface membrane of intact lymphocytes was labelled with d‐biotinyl‐e‐aminocaproic acid‐N‐hydroxysuccinimide ester (biotin‐7‐NHS) as described by the manufacturer (Roche Diagnostics GmbH, Germany). For immunoprecipitation, adherent cells were biotinylated, released by a cell scraper and then immunoprecipitated. Cells in suspension and non‐adherent cells were biotinylated and immunoprecipitated directly. The reaction was stopped with 75 μl stop solution per tube after incubation for 15 min at room temperature and centrifuged at 490 g for 10 min. The supernatant was discarded and 5 ml cold PBS was added to each tube followed by centrifugation at 490 g for 10 min. The cells were lysed in 1 ml lysis buffer (50 mm core buffer, 150 mm NaCl, 0·1 mg/ml PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1% Nonidet P‐40 and 0·5% sodium deoxycholate) and incubated for 30 min on ice. After incubation for 15 min the cells were resuspended and centrifuged at 12 000 g for 10 min at 4° and the supernatants were transferred to clean Eppendorf tubes.

Immunoprecipitation was essentially carried out with protein G agarose beads as described by the manufacturer (Roche). The supernatants were mixed with 1 μg antibody at 4° overnight followed by centrifugation at 12 000 g at 4° for 20 seconds. Subsequently, the supernatants were discarded and the beads were resuspended in 1 ml washing buffer, and centrifuged again at 12 000 g at 4° for 20 seconds, the same procedure was repeated twice. After washing, 20 μl reducing buffer (2×, containing 0·15 g dithiothreitol in 5 ml immunoprecipitation buffer (150 mM NaCl, 10 mM Tris‐HCL pH 7.4)) was mixed with the beads and heated at 95° for 4 min and subsequently centrifuged at 7000 g for 1 min to spin down the beads and the proteins were separated on SDS–PAGE gels. Proteins were transferred to the Hybond ECL membrane (Amersham, Chalfont St Giles, UK) and detected using the BMC chemiluminescence blotting kit (Roche).