Figure 6: DJ-1 stabilizes the cellular levels of 20S proteasome substrates.
From: The Parkinson’s-associated protein DJ-1 regulates the 20S proteasome
HeLa and A31N-ts20 BALB/C cells were transiently transfected to silence (a–c) or overexpress DJ-1WT and its mutational variant DJ-1C106A (d,e), with (c,e) or without (a,b,d) FLAG-p53. Forty-eight hours after transfection, A31N-ts20 BALB/C cells were moved to 39 °C. The levels of the different proteins were examined by western blot analyses 72 h post transfection. As controls, non-targeting siRNA (NT) (a–c) or the fluorescent protein Cerulean (CFP) (d,e) were used. The results indicate that silencing DJ-1 or overexpressing the dominant negative DJ-1C106A, reduce the cellular levels of 20S proteasome substrates p53 and α-synuclein. (f) To evaluate the degradation rate of α-synuclein in cells, depleted of DJ-1, a CHX chase was performed, using A31N-ts20 BALB/c cells transfected with siRNA against DJ-1 or non-targeting siRNA. Forty-eight hours after transfection, the cells were moved to 39 °C and 72 h post transfection, CHX was added. Cell extracts were prepared at the indicated times following the drug addition, analysed by immunoblotting and quantified by image analysis. Error bars represent s.e. (g) The impact of DJ-1 knockdown on the stability of MDM2, which is degraded in an ubiquitin-dependent manner by the 26S proteasome, was examined in A31N-ts20 BALB/C cells. After transfection, the cells were grown at 32 °C. Western blot analyses were performed 72 h post transfection with DJ-1 siRNA or non-targeting siRNA (NT). GAPDH, tubulin (Tub) and Ponceau S-staining served as loading controls. The average level of DJ-1 in silenced HeLa and A31N-ts20 cells, relative to its level in non-silenced cells was 22%±4% (n=5) and 23%±4% (n=21), respectively. Percent expression±s.e. of the different proteins, in DJ-1-silenced cells (a–c,g) or in cells overexpressing the mutational variant DJ-1C106A (d,e), relative to control cells was calculated. Values are shown under the name of the proteins tested (α-synuclein in a,b,d, FLAG-p53 in c,e and MDM2 in g) of relevant blots. All experiments were repeated between three and seven times.
