Figure 6. Cell surface interaction of Cdc37 and HSP90.

A, Membrane protein fractions from MDA-MB 453 and MDA-MB 231 cells (control) were immunoprecipitaded with anti-Cdc37 antibody. Analysis of bound proteins by Western blot with anti HSP90 antibody and anti HER-2 and anti –EGFR antibodies respectively, revealed that Cdc37 interacts not only with HSP90 but also with both ErbB receptors studied. Presence of 200 µg/ml of the cell impermeable anti-Cdc37 for 16 h in the culture medium of the two cell lines followed by immunoprecipitation and Western blot analysis as above revealed that the anti-Cdc37 antibody disrupts the association of Cdc37 with HSP90 and the ErbB receptors in the two cell lines. B, MDA-MB 453 and MDA-MB 231 cells were treated or not with 200 µg/ml mAb 4C5 for 16 hours. Membrane protein fractions derived from these cultures were immunoprecipitaded with anti-Cdc37 followed by Western blot analysis using anti-HSP90 and anti-HER2 and anti EGFR antibodies respectively. C, Membrane protein fractions from MDA-MB 231 cells cultured in the absence or presence of 200 µg/ml of mAb 4C5 for 16 h were immunoprecipitated with the anti-EGFR antibody. Analysis of bound proteins by western blot with anti-HSP90 antibody revealed that surface HSP90 interacts with EGFR and that this interaction is disrupted by mAb 4C5. Irrelevant IgGs served as negative control for all the above experiments. D, Densitometry quantification of the observed reduced interactions in the presence of anti-Cdc37 in the culture medium of MDA-MB 453 and MDA-MB 231 cells resulted in a 33% and 59% decrease with HSP90 and HER-2 respectively regarding the MDA-MB 453 cells and in a 56% and 47% decrease with HSP90 and EGFR respectively regarding the MDA-MB 231 cells. Presence of mAb 4C5 in the culture medium of MDA-MB 453 and MDA-MB 231 cells resulted in a 60% and 58% decrease with HSP90 and HER-2 respectively regarding the MDA-MB 453 cells and in a 88% and 70% decrease with HSP90 and EGFR respectively regarding the MDA-MB 231 cells.