DLK affects the formation of the JIP1–JIP3–kinesin-1 ternary complex. (A). Lysates of Neuro2a cells expressing TAP-JIP1 and T7-DLK-WT, T7-DLK-KR or pUC8 were precipitated with SA and analyzed by WB with the indicated antibodies. SA, proteins bound to streptavidin sepharose. (B). Quantification of the binding of JIP3 and kinesin-1 to TAP-JIP1 in (A). The extent of binding was normalized to the amount of precipitated TAP-JIP1. *: p < 0.036 compared with Empty. Error bars indicate ± SEM. n = 4. (C). Differentiated RFP-JIP1-Neuro2a cells were transfected with expression vectors for T7-tagged DLK-WT, T7-DLK-KR, or pUC8 (empty vector). T7-DLK was detected by indirect immunofluorescence microscopy using anti-T7 antibody. Arrowheads indicate the neurite tips of transfected cells identified by phase contrast microscopy. Scale bar = 20 μm. (D). Quantification of the relative fluorescence of RFP-JIP1 at the neurite tip. *: p < 0.001. Error bars indicate ± SEM. n = 100 neurites for each construct.
Satake et al. BMC Cell Biology 2013 14:12 doi:10.1186/1471-2121-14-12