MAb4C5 prevents MMP2 and MMP9 activation and disrupts their interaction with both the α and the β isoform of extracellular HSP90. (A) Metalloproteinases derived from the concentrated supernatants of control and mAb 4C5 treated cultures of MDAMB453 cells were isolated and analyzed by zymography, as described in Methods. Proteolysis was detected as a white zone in a dark field. Metalloproteinases previously isolated as described in Methods were used as markers. Activated MMP2 and MMP9 are absent in the mAb 4C5 treated cultures. (B) Proteins derived from the concentrated supernatants of control and mAb 4C5 treated cultures of MDAMB453 cells, prepared as described in Methods, were analyzed by Western blot using anti-MMP2 and anti-MMP9 antibodies. Presence of mAb 4C5 in the culture medium inhibits activation of both metalloproteinases. (C) Proteins from the above described supernatants were immunoprecipitated with anti-MMP2 and anti-MMP9 antibodies and bound proteins were analyzed by Western blot with anti-HSP90α and anti-HSP90β antibodies. MAb 4C5 disrupts the association of both isoforms of HSP90 with the two metalloproteinases, since anti-MMP2 and anti-MMP9 antibodies did not co-immunoprecipitate any detectable levels of HSP90 in the mAb 4C5 treated cultures, when compared with controls. Western blot analysis of the MMP2 and MMP9 immunoprecipitants, using the corresponding antibodies was performed as positive controls and irrelevant IgGs were used as negative controls. IP immunoprecipitation, WB Western blot.
Stellas et al. BMC Cell Biology 2010 11:51 doi:10.1186/1471-2121-11-51