Figure 2

Identification of JIP3 as a major PTB domain-dependent binding protein of JIP1. (A). Neuro2a cells transiently transfected with either TAP-JIP1 or control vector were used for pull-down experiments with streptavidin sepharose (SA). The JIP1 associated polypeptides were visualized by silver staining. (B). Co-precipitation of GFP-JIP1 constructs and endogenous JIP3 in Neuro2a cells. The same samples as shown in Fig. 1B were analyzed by WB with the indicated antibodies. (C). Lysates of HEK293T cells expressing TAP-JIP1, V5-tagged kinesin light chain (V5-KLC), V5-KHC, GFP-JIP3, DLK, APP, or GFP alone were precipitated with SA and analyzed by WB with the indicated antibodies. Input, cell lysate used for the immunoprecipitation assay. SA, proteins bound to streptavidin sepharose. (D). Quantification of kinesin-1 (V5-KLC) binding to TAP-JIP1 in (C). Kinesin-1 (V5-KLC) binding was normalized to the amount of precipitated TAP-JIP1. *: p < 0.003 compared with GFP. Error bars indicate ± SEM. n > 3. (E). An E17 mouse brain extract was immunoprecipitated with anti-JIP1 antibody. Precipitates were analyzed by WB with the indicated antibodies.