FIGURE 7.

Analysis of recombinant ASL activities. Shown is ASL enzymatic activity analysis in non-transfected or (co-)transfected 293T cell extracts. Each 4.8–14 μg of total protein of cell extracts was used for the enzyme assay. The residual ASL activities are represented as a percentage of ASL-WT activity (percentage of ASL-WT or of ASL-WT with EV). A, 293T cells were transiently transfected with 7 μg of P-WT (wt), P-ex2del (ex2del), or P-ex7del (ex7del), respectively. NT, non-transfected. Non-transfected cells and those transfected with EV served as negative control. B, 293T cells were transiently co-transfected with 10.5 μg of total plasmids at a ratio of 1:1 (each 3.5 μg) of P-WT to P-ex2del or P-ex7del plasmid, respectively. C, 293T cells were transiently co-transfected with 7 μg of total plasmids at a ratio of 1:1 (each 3.5 μg) of P-WT to P-ex2del or P-ex7del and of P-E189G to P-ex2del or P-ex7del plasmid, respectively. D, ASL enzymatic activity in 293T cell extracts co-transfected with P-WT and P-ex2del at different ratios. 3–12 μg of cell extracts was used for ASL enzyme analysis depending on the amount of P-WT used for the co-transfections. For co-transfections, different ratios of ASL-WT to ex2del splice variant (1:1, 1:2, 1:5, 2:1, or 5:1) were used, respectively. The residual activity of each co-transfectant compared with ASL-WT under the same condition is indicated as a percentage of ASL-WT activity (percentage of ASL-WT co-transfected with EV to use identical amounts of total plasmids and of P-WT for all experiments). EV was used to set up the same amount of total plasmids for the co-transfections. Levels of significance are given as p values obtained by one-way analysis of variance using GraphPad software from triplicate measurements of at least three independent experiments, respectively. Differences were considered as significant if the p value was <0.05. Error bars, S.D.