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In situ cross-linking. This was performed using Dithiobis[succinimidylpropionate] (DSP) (Pierce Perbio Ltd.) as described previously [12]. Briefly, the biotinylated cell monolayers were incubated in PBS8, and DSP added to give the required concentration. Prior to detergent extraction the monolayers were extensively washed with PBS8 (supplemented with 2 mM lysine).
Immunoprecipitation. This was performed as described previously [4]. Briefly, cell lysates were prepared at 4 °C using either RIPA buffer (1% NP-40, 0·1% SDS, 150 mM NaCl, 1 mM EDTA, 2 mM PMSF, 2 mM lysine, 20 mM Tris–HCl, WEHI-539 7.5) or NP40 buffer (1% NP40, 150 mM NaCl, 1 mM EDTA, 2 mM lysine, 2 mM PMSF, 20 mM Tris–HCl, pH 7.5) and clarified by centrifugation (13,000g, 10 min 4 oC). Aliquots of the lysates were incubated with the relevant primary antibody, and the immune complexes were isolated with protein A–Sepharose. The immune complexes were then resuspended in boiling mix and analysed further.
Western blotting. The proteins were separated by SDS–PAGE, transferred by Western blotting on to PVDF membranes, and the membranes incubated in blocking buffer (1% Marvel?, 0.05% Tween in PBS). The membranes were then either incubated with the relevant primary antibody and a suitable secondary antibody conjugated to horse radish peroxidases (HRP), or alternatively, incubated with streptavidin conjugated to HRP (Amersham) for the detection of biotinylated protein species. Protein bands were visualised using the ECL protein detection system (Amersham). Apparent molecular masses were estimated using Kaleidoscope (Biorad) prestained protein markers in the molecular weight range, 14–200 kDa.