Figure 2.

RAB6A Is More Sensitive Than RAB27A to Assess the Biological Activity of Human REP1 following rAAV2/2 Transduction of HEK293 Cells

(A) The HEK293 cells were transduced with increasing MOI of rAAV2/2-REP1 (100, 300, 1,000, 3,000, 10,000, 30,000, 100,000, and 300,000). Protein expression (human REP1 and β-actin) and biotin incorporation were detected in prenylation reaction products (20 μg) following SDS-PAGE and western blot analysis (representative image of three independent experiments). (B) Nonlinear regression plot of normalized REP1 (corrected for the corresponding actin levels) per rAAV2/2-REP1 (log gc/cell). Data were analyzed using a sigmoidal four-parameter fit (95% confidence interval; R² = 0.8625). Symbols are mean of 6 replicates ± SEM. C) Nonlinear regression plots of biotin incorporation per MOI of rAAV2-REP1 (log gc/cell). Data was analyzed using a sigmoidal four-parameter fit (95% confidence interval; R² = 0.8873 for RAB6A; R² = 0.8772 for RAB27A). Symbols represent the mean of three replicates ± SEM. RAB6A showed statistically significant higher incorporation of biotin than RAB27A at MOI 10,000 (**p = 0.01) and 30,000, 100,000, and 300,000 (****p ≤ 0.0002) (two-way ANOVA with Bonferroni’s multiple comparison test). (D) Linear regression plots of biotin incorporation in substrate corrected for the untransduced control against the normalized overexpressed REP1 for RAB6A (R² = 0.8959, Y = 18.82 × X + 0.4803) and RAB27A (R² = 0.533, Y = 6.569 × X + 0.9042).