Protein expression analysis in a panel of human breast cancer cell lines and effect of NVP-AUY922 on the HSP90-p23 complex in BT-474 cells. (a) The expression of HSP90 and specific proteins affected by HSP90 inhibition (Her3 [EGFR3], Her2 [ErbB2, EGFR2], phospho Her2 [pHer2], EGFR, AKT [PKB], phospho-AKT [pAkt], estrogen receptor-alpha [ERa], PI3K [p110α], PDK1, HSP70, Hsc70, Rb, pMEK1/2, pERK, Bax, Bcl-2, Bad, and Bcl-XL) was analyzed in seven human breast cancer cell lines (BT20, BT-474, MDA-MB-157 [MB-157], MDA-MB-231 [MB-231], MDA-MB-468 [MB-468], SKBr3, and MCF-7) by Western blot analysis. (b) The ERBB2-overexpressing and estrogen receptor-positive cell line BT-474 was chosen for studies of the kinetics and concentration-dependent dissociation of HSP90-p23 complexes and client proteins in the presence of NVP-AUY922 or tanespimycin (17-AAG). The amount of p23 associated with HSP90 was determined by immunoprecipitating p23 followed by immunoblotting for HSP90. The levels of ERBB2, AKT, phosphorylated AKT and β-tubulin were determined by immunoblotting. DMSO, dimethyl sulfoxide; HSP90, heat shock protein 90.