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. 2001 Mar 5;152(5):959–970. doi: 10.1083/jcb.152.5.959

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© 2001 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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A kinesin dominant negative construct causes mislocalization of expressed JIP-2 and JIP-3 proteins but not endogenous mitochondria, neurofilaments, or MTs. (A and B) CAD cells were transiently transfected with plasmids encoding the HA-tagged KLC TPRs together with Flag-tagged JIP-2 (A) or Flag-tagged JIP-3 (B). After differentiation, the expressed proteins were detected by indirect immunofluorescence microscopy using a mAb to the HA tag (left) and a polyclonal antibody to the Flag tag (right). Arrowheads denote tips of transfected cells; arrows denote tips of untransfected cells. (C–E) CAD cells were transiently transfected with a plasmid encoding the HA-tagged KLC TPRs. After differentiation, the expressed protein was detected by indirect immunofluorescence microscopy using a polyclonal antibody to the HA tag (left). Endogenous MTs, neurofilaments (NF), and mitochondria were detected with mAbs to tubulin (C, right) or neurofilaments H + M (D, right), or with MitoTracker (E, right). Note that in C, the cells were only allowed to differentiate for 12 h, so that MT organization could be assessed before the cell bodies rounded up.