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. Author manuscript; available in PMC: 2009 Jun 15.
Published in final edited form as: Cancer Res. 2008 Jun 15;68(12):4833–4842. doi: 10.1158/0008-5472.CAN-08-0644

Figure 6. Anti-Ac-K69 antibody inhibits hsp90α-dependent in vitro invasion by breast cancer cells.

Figure 6

(A) K/Q substitution at K69 promotes extracellular location of hsp90α. MB-231 cells transfected with either F-hsp90 or K69Q mutant were cultured under serum-free condition for 24 hours. Total extracellular and intracellular hsp90α were immunoprecipitated with anti-M2 conjugated beads and immunoblotted with anti-F antibody. The intensity of the bands was quantified using ImageQuant 5.2 software, and the ratio for intracellular to extracellular hsp90 is shown below the panel. Lane 1 is intracellular, and Lane 2 is extracellular. (B) Anti-Ac-K69 hsp90α antibody specifically recognizes acetylated form of both exogenous and endogenous hsp90α expressed in MB-231 cells. MB-231 cells were transfected with F-hsp90α followed by the treatment with LBH589. Immunoprecipitates of F-hsp90α with anti-M2 conjugated beads were immunoblotted with anti-K69-hsp90α antibody for the acetylation status and anti-F antibody for F-hsp90α expression. Cells transfected with empty vector followed the treatment with LBH589 served as control for specificity. Acetylation of endogenous hsp90α induced by LBH589 was also detected with anti-AcK69-hsp90α antibody (Lower panel). Immunoprecipitates of endogenous hsp90α with anti-hsp90α antibody from cell lysates of MB-231 cells treated with or without LBH589 were immunoblotted with either anti-AcK69-hsp90α or anti-hsp90α (rabbit) antibody. IgG served as the control for specificity of the immunoprecipitates. (C) LBH589 induces surface localization of acetylated hsp90α in MB-231 cells. Serum-starved MB-231 cells were treated with 40 nM LBH589 for 16 hours, followed by staining with anti-AcK69 hsp90α antibody and confocal microscopy. Cell cultured in RPMI with 10 % FBS and cells stained with rabbit IgG served as controls. (D) Inhibition of in vitro invasion by MB-231 cells by anti-AcK69 hsp90α antibody. Serum-starved MB-231 cells treated with 20 μg/mL anti-hsp90α or anti-AcK69 hsp90α antibody were used for determining in vitro matrigel invasion. Untreated cells, or cells treated with IgG were used as controls. Columns, average results of three independent experiments; bars, SD. *, p < 0.05.