Skip to main content
. Author manuscript; available in PMC: 2007 Jan 31.
Published in final edited form as: Cancer Res. 2005 Jun 15;65(12):5238–5247. doi: 10.1158/0008-5472.CAN-04-3804

Figure 2.

Figure 2

A, biophysical characterization of vesicles released by Colo−/Colo+ and CX−/CX+ carcinoma sublines. Sucrose density gradient centrifugation of Colo+ (left) and CX+ (right) derived vesicles was done on a sucrose density gradient by ultracentrifugation in a density range of 1.08 to 1.24 g/mL. Fractions were collected from top to bottom and analyzed after separation on a 10% SDS-PAGE by silver stain (top). As a marker for exosomes, acetylcholinesterase activity was measured in each individual fraction by standard colorimetric assay. Maximum protein amount and acetylcholinesterase activity were detected at a density of 1.17 g/mL. One representative experiment of three was shown. B, biochemical characterization of exosomes released by Colo−/Colo+ and CX−/CX+ carcinoma sublines. Comparative analysis of the protein composition in whole cell lysates (lys) and exosomal (exos) fractions of tumor sublines Colo−/Colo+ and CX−/CX+, as determined by silver staining (top) and Western blot analysis (bottom), using specific antibodies directed against tubulin, Bag-4, Hsp70, Hsc70, Rab-4, Rab-7, Rab-9, Rab-11, Grp94, and Calnexin. One representative silver stain and Western blot out of five was shown. C, surface of exosomes derived from Colo+ and CX+ carcinoma sublines are strongly positive for Hsp70/Bag-4. Exosomes derived from Colo−/Colo+ (left) and CX−/CX+ (right) carcinoma sublines were covalently bound to aldehyde/sulfate latex beads with a diameter of 4 μm. Exosome-coated beads were stained with antibodies directed against Hsp70 (cmHsp70.1, top, white histograms), Bag-4 (IMG-152, bottom, white histograms), and isotype-matched control antibodies (gray histograms) and analyzed by flow cytometry. Only the population containing single beads was gated and analyzed. Mean values of three independent experiments ± SE were summarized in Table 1B. D, immunoelectron microscopic view of the Hsp70 localization on the exosomal surface of CX− (top) and CX+ (bottom) cells at a magnification of ∼ ×60.000; scale bar, 50 nm. Localization of Hsp70 was visualized by 10 nm gold particles. No staining was found on exosomes incubated with an isotype-matched negative control antibody (data not shown).

HHS Vulnerability Disclosure