Figure 6.

Both synaptojanin and dynamin are unable to rescue the inhibitory effects of GST–endoSH3. (a) Permeabilized cells which had been preincubated with either GST–amph2 SH3^D36R (filled squares) or GST–endoSH3 (filled diamonds) for 5 min at 30°C were collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and increasing concentrations of dynamin for the ability to support B-SS-Tfn sequestration using the avidin inaccessibility assay. (b and c) Permeabilized cell membranes were preincubated in the presence or absence of GST–endoSH3 (0.1 mg/ml) for 5 min. They were then collected by centrifugation and assayed in the presence of ATP, cytosol (2.5 mg/ml), and synaptojanin (5 μg) as indicated for the ability to support avidin inaccessibility (b) or MesNa resistance (c) of B-SS-Tfn. Results are expresssed as the mean ± SD of two experiments each performed in duplicate. (d) Permeabilized membranes which had been preincubated in the presence of GST–endoSH3 and then subsequently incubated for 30 min in the presence of ATP and cytosol (lanes 1–3), plus dynamin (5 μg) (lane 2), plus synaptojanin (5 μg) (lane 3) were solubilized and electrophoresed on a 10% SDS gel, transferred to nitrocellulose, and probed for the presence of GST–endoSH3 using anti-GST antibodies.