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. Author manuscript; available in PMC: 2009 Feb 5.
Published in final edited form as: J Cell Physiol. 2008 Oct;217(1):250–260. doi: 10.1002/jcp.21500

Fig. 4.

Fig. 4

Sca-1 associates with Insulin Degrading Enzyme. A: Lysates from C2C12 myoblasts grown under differentiation conditions for 24 h were immunoprecipitated with anti-Sca-1 antibody (αSca-1) versus isotype control (IgG), and separated by SDS–polyacrylamide gel electrophoresis. Coomassie Brilliant blue staining identified aSca-1-dependent band at~110kDa protein, which was identified as IDE by LC–MS/MS analysis. B:Sca-1 was immunoprecipitated from differentiating C2C12 myoblasts, and immunoblot analysis showed that IDE (arrow) specifically co-precipitates with Sca-1 (αSca-1), compared to control immunoprecipitation with isotype control antibody (IgG). WCL, whole cell lysate. C: Immunoblot analysis as in (B) demonstrated Sca-1 immunoprecipitation. D: Details of GST-Sca-1 fusion construct in pETGEXT designed to exclude ER retention signal and GPI-anchor addition sequences. E: Empty vector (GST) and GST-Sca-1 were expressed in bacteria, purified, and used to pull down complexes from lysates of C2C12 myoblasts after 24 h of differentiation. Immunoblot analysis identified IDE in the pre-pulldown lysate (Pre), in lysate incubated with GST-Sca-1, but not in lysate incubated with GST alone.