Figure 3
From: Late-onset spastic ataxia phenotype in a patient with a homozygous DDHD2 mutation
PLA1 activity of DDHD2 mutants.
GST and GST-tagged DDHD2 WT and/or DDHD2 mutants were expressed in 293T cells and partially purified. Their PLA1 activities were measured using 32P-labeled DOPA as a substrate, as described in “Methods”. The reaction products were analyzed by TLC. (A): Representative results of TLC analysis. GST (lane 1), GST-DDHD2 WT (lane 2), a mixture of GST-DDHD2 WT and one of GST-DDHD2 mutants (p.Val220Phe (lane 3), p.Trp103Arg (lane 4) and p.Asp660His (lane 5)) and GST-DDHD2 mutants alone (p.Val220Phe (lane 6), p.Trp103Arg (lane 7) and p.Asp660His (lane 8)) were used. Positions of DOPA and LPA are indicated by arrows. The product LPA was clearly detected in the lane with GST-DDHD2 WT but not with GST alone. The amounts of LPA were markedly reduced in the cases of all the three mutants. (B): A representative image of Western blotting analysis. GST and GST fusion proteins used for the above PLA1 assay were analyzed by Western blotting using an anti-GST antibody. One-fifth of amount of each sample used in A was loaded. Lane numbers are the same as in A. (C): Comparison of PLA1 activity. The intensities of spots on a TLC plate were quantified using Multi Gauge V3.0 software (Fujifilm). The PLA1 activity (an amount (nmol) of LPA formed per min) was calculated from the intensities of LPA and DOPA spots. Data are shown as means ± S.D. from 3 or more independent experiments. Numbers in the graph are the same as in A. The asterisks indicate statistically significant differences between the DDHD2 mutants and control DDHD2 WT (asterisks placed above each bracket), or between GST and each condition of DDHD2 (asterisks placed above each bar) (*P < 0.05, **P < 0.001 and ***P < 0.0005, Student's t-test).
