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. Author manuscript; available in PMC: 2015 Apr 15.
Published in final edited form as: Biochem J. 2011 Aug 15;438(1):39–51. doi: 10.1042/BJ20110129

Figure 5. Conformation-sensitive antibodies used to detect pro-uPA or uPA on the cell surface.

Figure 5

A, Western blot analysis of conditioned media from PC-hi/diss cells grown in serum-free medium or in medium supplemented with 0.1% chicken serum with or without 0.1 TUI/ml aprotinin. The proteins were separated by SDS-PAGE under reducing conditions, transferred to a membrane support and probed with a polyclonal anti-uPA antibody. Pro-uPA is detected as one band migrating at Mr ~54,000, whereas active uPA is separated into two chains, migrating at Mr~34,000 (the catalytic domain, B-chain) and Mr~20,000 (the amino-terminal fragment, A-chain). *, non-specific band. B, Immunofluorescence analysis of cell surface-associated pro-uPA and active uPA. PC-hi/diss cells were grown in serum-free medium or in the presence of 0.1 % chicken serum with or without 0.1 TIU/ml aprotinin. After two days of incubation, the cells were fixed and stained without permeabilisation with mAb-112 or mAb-12E6B10. Anti-CD44 mAb-29-7 and mouse IgG were used as positive and negative controls, respectively. FITC-conjugated goat anti-mouse antibody was used to detect bound antibodies. Cell nuclei were stained with DAPI.