Figure 1.

Both the α and the β isoforms of HSP90 are secreted by MDAMB453 cells and interact with MMP2 and MMP9 metalloproteinases. (A) Cell lysates were immunoblotted with mAb 4C5 recognizing both isoforms of HSP90 and serving as positive control. Concentrated supernatants derived from the cell cultures as described in Methods, were analyzed by Western blot using anti HSP90 α and anti HSP90 β antibodies and mAb 4C5. Anti beta actin antibody was used in both fractions. Secretion of the two isoforms of HSP90 was detected in the supernatant derived from MDAMB453 cells. The absence of beta actin in the supernatant demonstrates that there is no contamination of this fraction with the intracellular components of cells during the experimental procedure. (B) Western blot analysis of concentrated supernatants derived from MDAMB453 cell cultures, using anti-MMP2 and anti-MMP9 antibodies showed that both the pro-MMP2 and pro-MMP9 as well as their activated forms are present in the culture medium. Proteins derived from the culture supernatant were immunoprecipitated with anti-MMP2 and anti-MMP9 and bound proteins were analyzed by Western blot with anti-HSP90α and anti-HSP90β antibodies. Both isoforms of HSP90 interact with the two metalloproteinases. Irrelevant IgGs and anti-MMP2 or anti-MMP9 antibodies were used as negative and positive controls respectively. (C) Proteins derived from the MDAMB453 cell culture supernatant were immunoprecipitated with anti-HSP90α and anti-HSP90β antibodies and immunoprecipitants were analyzed by Western blot using the anti-MMP2 and anti-MMP9 antibodies. Irrelevant IgGs and anti-HSP90 α or anti-HSP90 β antibodies were used as negative and positive controls respectively. The reverse immunoprecipitation experiment showed that both isoforms of HSP90 interact with pro-MMP2 and pro-MMP9 and to a lesser extent with the activated forms of the two metalloproteinases. IP immunoprecipitation, WB Western blot.

Stellas et al. BMC Cell Biology 2010 11:51   doi:10.1186/1471-2121-11-51
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