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. 2010 May 31;189(5):829–841. doi: 10.1083/jcb.200912105

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© 2010 Pearse et al.

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Figure 5. — Prosaposin displays nonnative characteristics. (A) Wild-type (WT) and ugt1^−/− cells were pulse labeled for 15 min, chased for 30 min where indicated, lysed, and treated with 5 mM PEG-maleimide (PEG-Mal) for 30 min on ice. PEG-maleimide was quenched in DTT, and prosaposin was immunoprecipitated with saposin C antisera. Samples were resolved via 7.5% SDS-PAGE. (B) Wild-type and ugt1^−/− cells were pulse labeled for 15 min and chased for the indicated times. After cell lysis, the Triton X-100 (TX-100)–insoluble pellet was solubilized in 1% SDS and quenched with excess Triton X-100. Samples were then subjected to immunoprecipitation with saposin C antisera. Samples were resolved via 7.5% nonreducing SDS-PAGE. (C) Myc/His-tagged human prosaposin (hPsap) was transiently overexpressed in wild-type and ugt1^−/− cells, pulse labeled for 15 min, and chased for the indicated times. Human prosaposin was isolated from cell lysates (lys) and media with antisera recognizing the myc epitope and analyzed via 7.5% SDS-PAGE. (A–C) Molecular mass is indicated in kilodaltons. (D) Quantifications of C using n = 3 and mean ± SD (error bars).