Figure 8

RNF182 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.