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. 2007 Jan 3;27(1):15–26. doi: 10.1523/JNEUROSCI.3826-06.2006

Figure 2.

Figure 2.

Complex formation with DISC1, Kinesin-1, and NUDEL in vitro and in vivo. A, An in vitro binding assay was performed using purified recombinant proteins. Beads immobilized with GST alone, GST–KLC1, or GST–KIF5A were incubated with MBP alone or MBP–DISC1. Bound proteins were analyzed by immunoblotting with antibodies against MBP and GST. The lower bands represent degradation products of MBP–DISC1. B, Lysates from transfected COS7 cells expressing NUDEL–GFP, Myc–DISC1, and HA–KIF5A were used in an immunoprecipitation assay with antibody against HA. The expression levels of NUDEL–GFP, Myc–DISC1, or HA–KIF5A were almost equal in each lane (data not shown). Bound proteins were analyzed by immunoblotting with antibodies against GFP, Myc, and HA. The lower bands represent degradation products of HA–KIF5A in immunoblotting with antibody against HA. C, D, Mapping of the regions in KIF5A or DISC1 required for binding to DISC1 (C) or KIF5A (D), respectively. E, F, Lysates from differentiated PC12 cells were used in an immunoprecipitation assay with antibodies (Ab) against DISC1 (E) or KIF5A (F). Bound proteins and inputs of assay were analyzed by immunoblotting with antibodies against the indicated proteins. Aliquots of original samples (10% Input) and eluates (30%) were subjected to SDS-PAGE.