(A–D) To identify cell surface localization of Hsp90 (csHsp90) by flow cytometry, HeLa (A–C) and primary dermal microvascular endothelial cells (pDMVEC) (D) were incubated with antibodies recognizing either the C-terminus (A), N-terminus (B and D), or an uncharacterized epitope (C) of the alpha isoform of Hsp90 followed by secondary antibodies conjugated to ALEXA-488 (black histograms). For controls, cells were incubated with secondary antibodies alone (gray histograms). (E–G) To validate these results, HeLa cells incubated with secondary antibodies conjugated to ALEXA-488 (E), anti-C-terminal Hsp90 Ab and secondary antibodies (F), or anti-N-terminal Hsp90 Ab and secondary antibodies (G) were examined by fluorescence microscopy. Original magnification × 60. Data shown represent one of three independent experiments.