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. 2010 Jul 12;190(1):129–141. doi: 10.1083/jcb.200912087

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© 2010 Kuang et al.

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Figure 5. — SPSB2 induces iNOS ubiquitination in vitro. (A) An in vitro ubiquitination assay was performed using recombinant E1, E2, and E3 ligase components and macrophage lysates as a source of iNOS. Excess free iNOS peptide was added as indicated. The reaction mixture was then separated by SDS-PAGE and analyzed by Western blotting with anti-iNOS antibody. Coomassie blue stain of the gel is shown in Fig. S2. (B) 293T cells were transiently transfected with cDNA expressing Flag-tagged SPSB2, lysed, and mixed with iNOS-expressing macrophage lysates containing increasing amounts of free iNOS peptide. Anti-Flag immunoprecipitates were then assessed for iNOS interaction by SDS-PAGE and Western blotting with anti-iNOS antibody. IP, immunoprecipitation.