Figure 4

Kalirin-IgFn domain disrupts dynamin self-assembly. A. Dynamin purified from adult rat brain was fractionated by SDS-PAGE and visualized with Coomassie brilliant blue: Bound, aliquot of washed GST-amphiphysin2-SH3 beads incubated with rat brain cytosol; Eluted, aliquot of protein eluted by high salt/lower pH buffer. B. Purified dynamin (2.5 μg) was incubated on ice with or without purified GST (5 μg) or purified GST-IgFn (5 μg) for 1 h before exposure to liposomes, then incubated with freshly prepared liposomes for 10 min at room temperature; the control lacked liposomes. Liposomes were pelleted and supernatants and pellets were fractionated by SDS-PAGE; proteins were visualized by Coomassie Brilliant Blue staining the PVDF membrane. Supernatant lanes tend to be wider because of the glycerol-containing sample buffer used to prepare these more dilute samples. C. Purified GST-IgFn domain (5 μg) was incubated with or without liposomes (composed of 0.1 mM PIP2 and 0.9 mM brain polar lipids), without dynamin; Coomassie Brilliant Blue staining is shown. D. Coomassie stained dynamin in the supernatant and pellet was quantified using Gene Tools (Syngene, Frederick, MD). For each experiment (n = 4), dynamin in the "liposome only" pellet was set to 100%; the amount of dynamin in the other pellets was normalized to this value. Error bars are standard error of the mean; p values, two-tailed student T-test.