Research Paper Volume 10, Issue 12 pp 3821—3833

Figure 1. Caloric restriction abrogates α-synuclein (SNCA)-induced toxicity by upregulating ubiquitin-proteasome system activity. (A) Chronological lifespan (CLS) and SNCA levels of stationary wild type cells harbouring the vector control or expressing the human SNCA grown under regular (2% glucose) or CR (0.5% glucose) conditions. (B) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. (C) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UB^G76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (D) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. (E) Ubiquitination profile determined by Western blotting using an anti-mono and polyubiquitination antibody. (F) Graphical representation of the intensity of total UB/Pgk1 obtained by densitometric analysis. (G) RPN4 and (H) RPN5 mRNA relative expression levels. Three reference genes (ACT1-actin, PDA1-alpha subunit of pyruvate dehydrogenase and TDH2-isoform 2 of glyceraldehyde-3-phosphate dehydrogenase) were used as internal standards and for the normalization of mRNA expression levels. Significance was determined by two-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) between cells grown under regular or CR conditions expressing vector control or SNCA. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).