FIG. 2.

Inhibition of nucleotide exchange on Rab11 in HD cells. One hundred micrograms of total membranes from primary control and HD fibroblasts was extracted with 1% Triton X-100 and centrifuged. The extracted supernatants were used to catalyze GDP release from [³H]GDP-GST-Rab11 and [³H]GDP-Rab5-His₆ at 30°C for 30 min (a) or [³H]GTP uptake into GDP-GST-Rab11 at 30°C for indicated times (b). [³H]GDP-GST-Rab11, [³H]GDP-Rab5-His₆, or [³H]GTP-GST-Rab11 on beads was collected by centrifugation, washed in cold washing buffer, and measured by scintillation counting. Data for panel a are represented as mean percentages ± SD for retaining [³H]GDP (n = 3; *, P < 0.01 by the Student t test). n/s, no significance. In panel b, the cpm value for each time point from each experiment was converted into picomoles, and the mean values in picomoles ± SD of [³H]GTP were graphed (n = 3; *, P < 0.01, determined by the Student t test).