Figure 8From: A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradationRNF182 targets ATP6V0C for proteosome degradation. N2a cells were transiently transfected with empty pEGFP-N1 or pCMV-Tag1 vector alone, pRNF182*EGFP or pCMV-Tag1-ATP6V0C alone, or pRNF182*EGFP and pCMV-Tag1-ATP6V0C simultaneously. Cells were collected for Trypan Blue exclusion assay as well as total RNA and protein extractions 24 h after transfection or treated with 30 μM MG132 for 8 h prior to total RNA and protein extractions. A. Over-expression of ATP6V0C in RNF182 transfected cells did not change the percentage of cell death caused by RNF182 over-expression. This figure shows the percentage of cell death before and after transfection. Bars represent the percentage of cell death in the population (mean ± SEM from 3 independent experiments performed in duplicate). Asterisks indicate a significant difference (ρ < 0.05; ANOVA, followed by Bonferronic test). B. Western and semi-quantitative PCR analyses of ATP6V0C and RNF182 protein and mRNA levels before and after transfection. IB: indicates primary anti-body used for immmunoblotting. β-actin was used as a loading control for both Western and PCR analyses. In: lane1 – transfection with empty pEGFP-N1 vector, lane 2 – transfection with pRNF182*EGFP, lane 3 – transfection with empty pCMV-Tag1 vector, lane 4 – transfecton with pCMV-Tag1-ATP6V0C, lane 5 – transfection with both pRNF182*EGFP and pCMV-Tag1-ATP6V0C. C. Western analysis of changes of ATP6V0C and RNF182 levels before and after transfection followed by MG132 treatment. IB: indicates primary anti-body used for immmunoblotting. Ubn: polyubiquitinated protein. Arrow indicates non-specific bands caused by anti-flag antibody. Western blotting of β-actin was used as a loading control.Back to article page