JIP1 binds directly to the p150^Glued subunit of dynactin. (A) Endogenous JIP1 and p150^Glued coimmunoprecipitate from mouse brain homogenate. (B) JIP1 binds to the C-terminal p150^Glued-CBD. Lysates from COS7 cells cotransfected with myc-JIP1 and FLAG-p150^Glued fragments were immunoprecipitated with an anti-FLAG antibody. JIP1 coimmunoprecipitated with C-terminal p150^Glued[880–1278], but not with N-terminal p150^Glued[1–880]. (C) JIP1 binds directly to C-terminal p150^Glued. When applied to His-Bind resin bound to His-JIP1, MBP-p150^Glued[1049–1278] selectively bound whereas MBP did not. (D) Summary of p150^Glued and JIP1 mapping results. (E) A diverse set of cargo adaptors binds to p150^Glued-CBD. (F) SNX6 and JIP1 bind competitively to p150^Glued. Lysates from COS7 cells transfected with fixed amounts of FLAG-p150 and myc-JIP1 DNA and progressively increasing amounts of GFP-SNX6 DNA were immunoprecipitated with anti-FLAG antibody. FLAG-p150 coimmunoprecipitates predominantly with myc-JIP1 in the low GFP-SNX6 condition, but mostly with GFP-SNX6 in the high GFP-SNX6 condition. (G) C-terminal JIP1 binds to p150^Glued. Lysates from COS7 cells cotransfected with FLAG-p150^Glued and myc-JIP1 fragments were immunoprecipitated with an anti-myc antibody. p150^Glued coimmunoprecipitated with both myc-JIP1[441–565] and myc-JIP1[554–711]; the presence of both these domains in JIP1 (myc-JIP1[441–711]) did not strengthen the interaction. Asterisk shows antibody light chain bands.