Figure 3

JIP1 requires JIP3 for binding to kinesin-1 in Neuro2a cells. (A). Lysates prepared from RFP-JIP1-Neuro2a cells expressing shRNA targeting JIP3 (shJIP3) or non-silencing control (NS) shRNA were immunoprecipitated with anti-RFP antibody and analyzed by WB with the indicated antibodies. Input, cell lysate used for the immunoprecipitation assay. IP:RFP, immunoprecipitated proteins. (B). Quantification of kinesin-1 binding by RFP-JIP1 in (A). Kinesin-1 binding was normalized to the amount of precipitated RFP-JIP1. *: p = 0.004 compared with NS. Error bars indicate ± SEM. n = 3. (C). Differentiated RFP-JIP1-Neuro2a cells were transfected with shRNA vectors (NS or shJIP3) containing a GFP expression cassette. Arrowheads indicate the neurite tips of transfected cells. Scale bar = 20 μm. (D). Quantification of the relative fluorescence of RFP-JIP1 and GFP in the same neurite tip. *: p < 0.001. Error bars indicate ± SEM. n = 200 neurites for each construct.