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. 2014 Dec 15;25(25):4024–4033. doi: 10.1091/mbc.E14-06-1167

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© 2014 Snider et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — Molecular and functional characteristics of CD73S. (A) Coimmunofluorescence analysis of the proliferation marker Ki67 (red) and Flag-CD73S/L (green) in transfected HepG2 cells. Blue, 4′,6-diamidino-2-phenylindole (DAPI); bar, 20 μm. (B) Quantification of Ki67 immunofluorescence staining in CD73-expressing cells relative to neighbor untransfected cells (30 CD73⁺ cells and 150 CD73⁻ untransfected cells were counted per condition). ****p < 0.0001, unpaired t test. (C) Measurement of 5′-nucleotidase activity in transfected HEK293T cell lysates. Samples expressing equal protein levels (bottom) were analyzed in triplicate. (D) Treatment of CD73-transfected HEK293T cell lysates with peptide-N-glycosidase F results in similar deglycosylation of both isoforms. (E) Immunofluorescence-based localization of Flag-CD73L or Flag-CD73S in primary mouse hepatocytes. Blue, DAPI; bar, 20 μm. (F) Flag immunoblot of Flag-CD73L– and -CD73S–expressing HEK293T total cell lysates analyzed under reducing (+β-mercaptoethanol, βME) or nonreducing (–βME) conditions, showing that CD73S does not dimerize. (G) Coomassie-stained gel of Flag immunoprecipitates of the nonreduced samples shown in F. Asterisk denotes a unique protein present in CD73S immunoprecipitates, identified by mass spectrometry as calnexin. (H) Biochemical validation that CD73S coimmunoprecipitates with calnexin.