Fig. 1. The role of CD82 in the dimerization of ErbB receptors. (A) Expression of ErbB receptors in HB2/ZEO and HB2/CD82 cells. Protein lysates (10 µg) were resolved in 8% SDS-PAGE, transferred to the nitrocellulose membrane and probed with the anti-EGFR (mAb-15), anti-ErbB2 (mAb-1) and anti-ErbB3 (C-17) Abs. (B) Cells were incubated with ¹²⁵I-EGF (or ¹²⁵I-TGF) at 4°C for 2 hours. The unbound ligand was removed and the cells were treated with 0.5 mM BS³ for 1 hour at 4°C. The complexes were purified as described in the Materials and Methods and resolved in 6% SDS-PAGE. The Abs used were: polyclonal 1005, anti-EGFR; mAb-11, anti-ErbB2; polyclonal C-17, anti-ErbB3. ErbB3 in the immunoprecipitates was probed with the mAb HER-3 Ab-6. (C) 0.5 µg of rs-CD82 was immunoprecipitated using a panel of anti-CD82 mAbs (lanes 2-5) or a negative control (187.1) mAb. 0.2 µg of purified rs-CD82 was used as a positive control (lane 1). The immunoprecipitates were resolved in 10% SDS-PAGE transferred to the membrane and probed with the anti-CD82 mAb (C33). In the additional experiments we found that denatured rs-CD82 is not recognised by any of the anti-CD82 mAbs (results are not shown). (D) The immunoprecipitation analysis was carried out as described in A except that cells were pre-incubated with the recombinant soluble (rs) proteins prior to (1 hour at 4°C) and during binding of ¹²⁵IEGF. The experiments with HB2/ZEO and HB2/CD82 cells were done in parallel. Both gels were exposed for 14 days.

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