Figure 1

The IgFn domain of Kalirin12 interacts with dynamin. A. The domains of Kalirin12 are shown: Dbl homology (DH); pleckstrin homology (PH); Src homology 3 (SH3); immunoglobulin (Ig); fibronectin III (Fn); the alternate C-terminus of Kalirin7 is shown. The specificities of the spectrin-directed (Kal) and Kalirin12 antisera are indicated. B. Cytosolic (Cyt) and solubilized organellar (Org) fractions (5 mg protein) from adult rat cortex were incubated with GST-IgFn beads; homogenization buffer (BLK) was analyzed as a control. Proteins bound to the beads were fractionated by SDS-PAGE, transferred to PVDF membranes and visualized with Aurodye Forte. The 100 kDa band bound specifically to GST-IgFn was sequenced (boxed). C. The domain structure of dynamin is illustrated: pleckstrin homology (PH); GTPase effector domain (GED); proline-rich domain (PRD). The approximate locations of the five tryptic peptides identified by MS-analysis are indicated. D. Adult cortical cytosol (2.0 mg protein) was incubated with GST-IgFn beads. Beads were washed and bound proteins eluted. Eluate equivalent to 200 μg of lysate protein was fractionated; input (20 μg protein) was analyzed for comparison. In addition to the pan-dynamin (Dyn) antibody, dynamin1 (Dyn1), 2 (Dyn2) and 3 (Dyn3) antibodies were used. E. Total dynamin, dynamins1, 2 and 3 and Kalirin12 (C-terminal antibody; Fig. 1A) were visualized in SDS lysates (20 μg protein) prepared from the tissues and cells indicated: E18, embryonic day 18 rat brain; P1, postnatal day 1 rat cortex (Ctx); adult rat cortex; PC12 cells.