Figure 4.

NDRG1 mediates SGK-1 suppression of Smad2/3. (A) C2C12 cells were treated with TGF-β1, and interactions between SGK-1 and Smad2/3 were determined by immunoprecipitation with anti–SGK-1 antibodies. The precipitated proteins were detected by Western blot using indicated antibodies. (B) Phosphorylation of NDRG1 was determined in C2C12 cells infected with control vector or AdSGK-1. (C) The interactions between NDGR1 and Smad2/3 were determined by immunoprecipitation of C2C12 cells with anti-NDRG1 antibodies. C2C12 cells, infected with AdGFP or AdSGK-1, were treated with TGF-β1 for 1 hour, and the interaction of Smad2/3 with NDRG1 was detected. (D) NDRG1 knockdown increases TGF-β1–induced Smad2/3 phosphorylation. Cells were treated with NDRG1 siRNA for 72 hours and then exposed to TGF-β1 for 30 minutes. The phosphorylation of Smad2/3 was determined. (E and F) Knockdown of NDRG1 suppresses SGK-1–mediated inhibition of Smad2/3 phosphorylation and Atrogin1/MuRF1 expression. C2C12 cells were infected with control or NDRG1 siRNA in the presence or absence of AdSGK-1. TGF-β1–induced (E) Smad2/3 phosphorylation and (F) Atrogin1/MuRF1 expression were determined by Western blot and RT-PCR, respectively. Data represent three repeats of the experiments. AdGFP, GFP adenovirus; CTL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IB, immunoblot; IP, immunoprecipitation; siRNA, short interfering RNA.