Figure 2

Kalirin12 interacts with dynamin in vitro and in vivo. A. Lysates of pEAK Rapid and PC12 cells (20 μg protein) were blotted for total dynamin (pan), dynamin1 (Dyn1) or dynamin2 (Dyn2). B. pEAK Rapid cells were transiently transfected with pEAK vector encoding myc-Kalirin12 (myc-K12) or pEGFP-N2 (Vector). Extracts (100 μg protein) prepared 48 h later were immunoprecipitated with antibodies to Kalirin spectrin (Kal). Immunoprecipitation was verified using Myc antibody (upper) and co-immunoprecipitation was assessed with antibody specific for pan-dynamin (Dyn, lower); ratio of Input to Immunoprecipitated sample, 1:10. C. Cortical cytosol (Cort. Ext.; μg indicated below blots) was immunoprecipitated with Kalirin-spectrin antibody (Kal; upper) or dynamin antibody (Dyn; lower); controls included IgG or no antibody. Immunoprecipitated proteins were visualized with the same Kalirin antibody (upper) or dynamin2 antibody (lower). Co-immunoprecipitation was assessed using antisera to dynamin, dynamin1, dynamin2 or Kalirin12 as indicated; ratio of Input to Immunoprecipitated sample, 1:100. D. Primary cultures of cortical neurons were fixed 3 h after plating. Endogenous Kalirin12 was visualized using a rabbit polyclonal antibody for Kalirin12 and Alexa488-secondary (green). Endogenous dynamin was visualized using the pan-dynamin antibody (Dyn) and Cy3-secondary (red). S, soma; GC, growth cone; boxed regions are shown enlarged. White arrows, colocalized Kalirin12 and dynamin. Scale bar: 10 μm.