Figure 1.

A, expression of Hsp70 and Bag-4 on the plasma membrane of human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines. Nonpermeabilized, 7-AAD negatively gated, viable carcinoma sublines Colo−/Colo+ and CX−/CX+ with intact plasma membrane were stained either with Hsp70− (cmHsp70.1) or Bag-4 (IMG-152) specific antibodies and analyzed by flow cytometry. The histograms represent positively stained cells in white, corrected for nonspecific staining, using an isotype-matched control antibody marked in gray. Mean values ± SE of three independent were summarized in Table 1A. B, Hsp70 and Bag-4 were actively released in detergent-soluble vesicles by viable carcinoma sublines. The amount of Hsp70 protein released by tumor sublines Colo−/Colo+ and CX−/CX+ was quantified by ELISA. Cell-free supernatants of Colo×/Colo+ (top) and CX−/CX+ (bottom) cells, diluted either in PBS buffer (control, ●), or in buffer containing the detergent Triton X-100 (1% v/v, o), Lubrol WX (1% v/v, △), and Brij 98 (0.5% v/v, ▲) were analyzed. One representative kinetic of three independent experiments was illustrated.