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. 2014 Feb 4;5:3224. doi: 10.1038/ncomms4224

Figure 8. NRDc binds to PGC-1α and negatively regulates its transcriptional activity.

Figure 8

(a) COS7 cells were transfected with the indicated expression vectors, followed by immunoprecipitation of the co-transfected cell lysates with anti-FLAG (PGC-1α) antibody. Twenty percent of input was blotted with anti-V5 (NRDc) in the bottom lane. Full blots are presented in Supplementary Fig. 9. (b) Pull-down assay using recombinant mouse NRDc and GST fusion proteins containing the first 400 amino acids of PGC-1α immobilized on glutathione-Sepharose beads. After the incubation, the beads were boiled and the eluted proteins were stained with Coomassie (left panel) and blotted with anti-NRDc (right upper panel) or anti-PGC-1α (right lower panel) antibody. Full blots are presented in Supplementary Fig. 9. (c) Luciferase reporter assay using COS7 cells transfected with Gal4-PGC-1α and different dose of NRDc. n=5. (d) Luciferase reporter assay using MEF−/− cells transfected with Gal4-PGC-1α and wild-type NRDc (WT) or enzymatically inactive NRDc (E>A). Protein expression of WT and E>A in cell lysates was confirmed by western blot with anti-NRDc antibody (bottom panel). Full blots are presented in Supplementary Fig. 9. n=5. All data represent means±s.e.m. *P<0.05, **P<0.01, ***P<0.001, NS, not significant (two-tailed Student’s t-test).