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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 6: — CD73S negatively regulates CD73L expression in a proteasome-dependent manner. (A) Measurement of 5′-nucleotidase activity in lysates from HEK293T cells transfected with equal amount of Flag-CD73L cDNA together with empty vector or Flag-CD73S. ****p < 0.0001, unpaired t test. (B) Measurement of CD73L (endogenous) and CD73S (transfected) mRNA in Huh-7 cells, showing that CD73S does not affect CD73L mRNA. (C) Determination of the effect of increasing levels of CD73S on CD73L dimer formation, which is detected under nonreducing conditions (–βME). (D) CD73 immunoblot of CD73-transfected HEK-293T lysates (±MG132). The same membrane under two exposure times from a triplicate experiment is shown. (E) Quantification of blot from D, showing that MG132 restores CD73L protein levels. ***p < 0.001, unpaired t test. (F) Reciprocal coimmunoprecipitation of CD73S/L-transfected HEK293T lysates, demonstrating that CD73S complexes with CD73L. Anti-V5 antibody was used as a negative control.