American Society of Hematology

Figure 1.

$Figure 1. Characterization and proteomic analysis of CLL plasma-derived exosomes. (A) Electron microscopy and NanoSight analysis of exosomes (Exo) purified from CLL plasmas. Comparative electron micrographs of MVs and exosomes from the same patient are depicted. Blue numbers indicate size of main peaks. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot characterization of plasma-derived exosomes. After protein quantification, exosomes were lysed in Laemmli buffer to perform SDS-PAGE and immunoblot. Different migration profiles in SDS-PAGE of exosome fractions and immunoblot with specific monoclonal antibodies against typical exosome proteins were used to validate the quality of our isolation technique. The proteins identified were tetraspanin CD9, heat shock protein HSP-70, and lysosomal marker LAMP-1. These were compared with the plasma of the same patient at the same disease time. Cytochrome C was also evaluated comparing whole-cell lysate (WCL) with plasma-derived exosomes of the same patient. (C) Subcellular localization of the proteins present in the plasma-derived exosomes purified of the different subgroups was analyzed by FunRich software. MWM, molecular-weight marker.$

Characterization and proteomic analysis of CLL plasma-derived exosomes. (A) Electron microscopy and NanoSight analysis of exosomes (Exo) purified from CLL plasmas. Comparative electron micrographs of MVs and exosomes from the same patient are depicted. Blue numbers indicate size of main peaks. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot characterization of plasma-derived exosomes. After protein quantification, exosomes were lysed in Laemmli buffer to perform SDS-PAGE and immunoblot. Different migration profiles in SDS-PAGE of exosome fractions and immunoblot with specific monoclonal antibodies against typical exosome proteins were used to validate the quality of our isolation technique. The proteins identified were tetraspanin CD9, heat shock protein HSP-70, and lysosomal marker LAMP-1. These were compared with the plasma of the same patient at the same disease time. Cytochrome C was also evaluated comparing whole-cell lysate (WCL) with plasma-derived exosomes of the same patient. (C) Subcellular localization of the proteins present in the plasma-derived exosomes purified of the different subgroups was analyzed by FunRich software. MWM, molecular-weight marker.

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