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. 1998 Jun 15;12(12):1775–1780. doi: 10.1101/gad.12.12.1775

Figure 2.

Figure 2

Figure 2

Figure 2

 mSiah2 targets N-CoR for proteasomal degradation. (A) mSiah2 blocks expression of N-CoR in 293T cells. Vectors expressing Flag–N-CoR (20 μg, lanes 2,3), Gal4–N-CoR(1–160) (20 μg, lanes 2,3), and PPARγ2 (10 μg, lanes 2,3) or control (lane 1) were transfected into 293T cells with mSiah2 expression vector (lane 3) or control. Gal4–N-CoR and PPARγ were cotransfected. For Flag–N-CoR detection, protein extracts (200 μg) were immunoblotted with monoclonal α-Flag antibody M2. For Gal4–N-CoR and PPARγ detection, 8 μg protein extracts were immunoblotted with either antibody to Gal4 or to PPARγ. (B) Half-life of N-CoR determined by pulse chase experiment. Results were quantitated in a PhosphorImager (Molecular Dynamics), normalized to t = 0 levels, and plotted in semilog format. The level of labeled N-CoR at t = 0 was reduced in mSiah2-transfected cells, attributable to increased degradation of protein synthesized during the time of the pulse. (C) 293T cells were transfected with expression vectors for indicated proteins and either no N-CoR/Gal4–N-CoR(1–160) (lane 1; top, 300 μg protein extract; bottom 29 μg extract), or Flag–N-CoR (300 μg extract, lanes 2–5, top) or Gal4–N-CoR(1–160) (29 μg extract, lanes 2–5, bottom), and treated with or without MG132 (20 μm). Cell extracts were analyzed by immunoblot analysis.