Fig. 1: Glucose deprivation induces ubiquitin-mediated degradation of Rheb.

A–C Time-dependent downregulation of Rheb protein level in response to glucose deprivation, but not to amino acids or serum deprivation. Huh7, HCT116, and EC109 cells were grown in EBSS (A), glucose-free, amino acids-free, serum-free medium (B) or treated with 2-DG (20 mM) (C) for the indicated time and subjected to immunoblotting (IB) analysis. D Decreased stability of Rheb during glucose deprivation. Cells were grown in full medium (DMEM high-glucose medium) and glucose-free medium containing 100 µg/ml CHX, and the protein level of Rheb was analyzed at the indicated time points. E Slowed degradation of Rheb during glucose deprivation by proteasome inhibitor MG132. Cells were grown in glucose-free medium containing CHX (100 µg/ml) + DMSO (0.1%) or 10 µM MG132, and protein levels of Rheb were analyzed at the indicated time points for IB analysis. F Increased levels of ubiquitinated Rheb upon MG132 treatment. Cells were treated with 10 µM MG132, and immunoprecipitation (IP) was performed using IgG or Rheb antibody (Ab). The polyubiquitination of Rheb (Rheb-Ub) was detected with Ub Ab. G Promotion of polyubiquitination of Rheb under glucose deprivation. Cells were cultured with or without glucose-free medium with 10 µM MG132, and IP was performed using IgG or Rheb Ab. H–J Enhanced protein stability of Rheb by MG132. Cells were treated with 10 µM MG132 and collected at the indicated time (H) or treated with MG132 at different concentrations (I) for IB analysis. J Cells were treated with 100 µg/ml CHX + DMSO or 10 µM MG132 and harvested at the indicated time for IB analysis. WCL, whole cell lysate. All data were representative of at least three independent experiments (n = 3).