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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Biochem J. 2008 Jul 15;413(2):251–259. doi: 10.1042/BJ20071496

Figure 5.

Figure 5

HAI-1 is rapidly endocytosed. MDCK-21 cells were grown on plastic until subconfluence and biotinylated with the cell membrane impermeable s-NHS-SS-biotin. After biotinylation, proteins were allowed to internalize for the times indicated at either 4°C or 37°C. Subsequently surface-exposed biotin was stripped by membrane impermeable reducing agent, glutathione (+), or reduction was omitted (-) and the cells were extracted. The cell extracts were streptavidin precipitated and the precipitates were analyzed by immunoblotting using the HAI-1 antibody. With immediate stripping of biotin after biotinylation, only a minor background of non-reduced HAI-1 could be detected. The bars represent the mean +/- SD (n=4) except for non-reduced samples where only a single determination is shown.