Alpha enolase

Western blot analysis was performed as described [30 (link)]. Fifteen μg total protein lysate was separated on 10% SDS-PAGE. Proteins were then transferred to a PVDF membrane (Amersham Biosciences, Piscataway, NJ). The membrane was blocked, incubated with primary antibodies overnight and secondary antibody for 1 h at room temperature. The membrane was developed with ECL Plus chemiluminescence reagent (Amersham Biosciences). The primary antibodies used in this study were against actin (Santa Cruz, CA), peripherin (Santa Cruz, CA), cytokeratin-8 (Abcam, MA), aldose reductase (Abcam, MA), alpha-enolase (Santa Cruz, CA), Grp75 (Cell Signaling Technology, MA), phosphoglycerate mutase (Santa Cruz, CA), F1 ATPase (Santa Cruz, CA), SCO2 (Santa Cruz, CA) and OGDH (Santa Cruz, CA).

Exosome-like vesicles were lysed in 40 μL of lysis buffer (Promega) containing 1 μL of proteinase inhibitor cocktail (Sigma). The total protein concentration was measured using a Bradford assay containing Coomassie Plus protein reagent (Bio-Rad Laboratories) according to the manufacturer’s specifications. Equivalent amounts of total lysate were subjected to SDS-PAGE using 10% polyacrylamide gels. Proteins were electroblotted to polyvinylidene difluoride membrane (Millipore). The membranes were then blocked and incubated in anti-Annexin A2 (rabbit polyclonal; Abcam), Alpha-enolase (mouse monoclonal; Santa Cruz), Anexin A1 (mouse monoclonal; Abcam), and EpCAM (mouse monoclonal; Abcam). Alkaline phosphatase–conjugated anti-mouse or anti-rabbit IgGs were used as secondary antibodies (Bio-Rad) for detection. Then the membranes were incubated with Western Blotting Detection Reagents (Bio-Rad) according to the manufacturer’s instructions and exposed to autoradiography film.