KIF5A-dependent βPIX targeting to the neurite tip (A, B). Representative images for the localization of βPIX (A) and Tiam1 (B) in differentiated PC12 cells. Kinesin constructs were GFP-tagged, and βPIX and Tiam1 were Myc-tagged. Two days after co-transfected cells were stained with anti-GFP and anti-Myc antibodies. Arrowheads at the line scan indicate neurite tips. Note specific KIF5A-dependent localization of βPIX at the neurite tip. (C) Quantification of the localization of βPIX and Tiam1 in (A, B). Accumulation of βPIX, and Tiam-1 at the neurite tip was quantified by line scan analysis using MetaMorph software. Student’s t-test. *P < 0.05, N = 30. (D) Representative images for the localization of GFP-tagged βPIX constructs in PC12 cells. (E) Quantification of the localization of βPIX constructs at the neurite tip in (D). Student’s t-test. **P < 0.01, N = 20. (F) Asso-ciation of βPIX constructs with KIF5A tail. PC12 cells were transfected with pEGFP-βPIX constructs for 2 days and were lysed and immunoprecipitated with anti-GFP antibody. Immunoprecipitates were incubated with purified His-tagged KIF5A tail followed by immunoblotting with anti-His antibody. Note that only βPIX-WT interacted with KIF5A tail (red arrowhead).
