Deacon SW , Serpinskaya AS , Vaughan PS , Lopez Fanarraga M , Vernos I , Vaughan KT , Gelfand VI .

GM52111 NIGMS NIH HHS , GM60560 NIGMS NIH HHS , T32 GM07283 NIGMS NIH HHS , R01 GM052111 NIGMS NIH HHS , R01 GM060560 NIGMS NIH HHS , T32 GM007283 NIGMS NIH HHS

	Figure 1. Kinesin II and the dynactin complex interact in melanophore extracts. (A) Kinesin II was precipitated from melanophore extracts with five different polyclonal (left) and one monoclonal (right) antibodies against the 95- and 85-kD subunits of kinesin II, respectively. Blots were probed with monoclonal anti-p150. Quantification of these blots shows that different kinesin II antibodies pull down 2–6% of the total p150 in the extract. (B) Dynactin was precipitated with polyclonal (left) or monoclonal (right) antibodies against p150. Blots were probed with monoclonal anti–kinesin II. Quantification of these blots shows that p150 antibodies pull down ∼1% of the total kinesin II in the extract. C indicates control samples precipitated either by normal rabbit IgG (left) or an unrelated monoclonal antibody (right).
	Figure 2. Direct interaction of p150 and XKAP. (A) 2.5 μg each of GST–C-XKAP and GST–N-XKAP were separated on a polyacrylamide gel and overlaid with 2 μg/ml p150Glued 1–811. Purified p150Glued 1–811 binds to GST–C-XKAP but not GST–N-XKAP. (B) Blot overlay assay using 2 μg/ml purified p150Glued 600–811. In this assay, p150Glued 600–811 showed robust binding to 1 μg each of purified DIC and GST–C-XKAP but not GST–N-XKAP. In A and B, the left panel is a Coomassie stain of purified proteins, and the right panel is blot overlay. (C) C-XKAP binds endogenous dynactin from melanophore extracts. GST–C-XKAP bound to glutathione agarose beads pulls p150 and dynamitin from cell extracts, whereas GST–N-XKAP does not.
	Figure 3. Kinesin II and dynein compete for binding to dynactin. (A) Polyclonal antibodies against DIC and the 95-kD subunit of kinesin II are each able to pull down p150 from melanophore extracts, but they do not pull down each other. Antibodies used for immunoprep are shown at the top. Antibodies used to probe the blot are indicated on the sides. (B) The ability of p150 to bind to purified DIC via blot overlay diminishes in the presence of increasing amounts of C-XKAP but not N-XKAP. Purified DIC was overlayed with myc- p150Glued 600–811 alone or in the presence of a 1:1 or 1:2 molar ratio of C-XKAP or N-XKAP. Blots were probed with an anti-myc antibody.

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