Tumor cell lines release BAG6 via the exosomal pathway. (A) Exosomes were isolated from MCF7 wt cells, from MCF7 cells stably overexpressing Smase2 (+Sm2) (left panel) and from MCF7+Sm2 cells upon control siRNA (AF488) transfection or siRNA-mediated knockdown of Smase2 (right panel) to detect BAG6 on exosomes by ELISA. Bar diagrams show mean and SEM (n = 3). See supplemental Figure 1 for reverse transcription-polymerase chain reaction to detect nSmase2 mRNA in wt cells or upon overexpression/down-regulation of nSmase2, respectively. (B) Western blots to detect BAG6, actin, and HSP70 in cell lysates, supernatant (SN) and purified exosomes (exo) from wt and +Sm2-MCF7 cells upon control and BAG6-transfection. (C) Western blot of 293T lysates with an anti-GFP antibody to detect expression of transfected GFP (control plasmid) or GFP-BAG6 fusion protein, consisting of GFP and BAG6 full length protein. Actin was used as a loading control. (D) FASC analysis of exosomes. The exosomes were purified from transfected cells and GFP-expressing exosomes were detected by GFP-fluorescence (left histogram): gray-filled histogram, GFP; black line, GFP-BAG6. Right histogram: anti-BAG6 staining of the same exosomes, detecting transfected and endogenous BAG6. Gray-filled histogram, istoype antibody; dashed line, GFP; thick black line, GFP-BAG6. GFP-expression data of 7 transfections were summarized in a bar diagram (mean and standard error of the mean).The means are significantly different (P = .0005, Wilcoxon signed-rank test).