Fig. 2. LMTK2 binds myosin VI in vitro and in vivo. (A) LMTK2 binds directly to purified myosin VI tail. Pull-down assays were performed using in vitro-translated LMTK2 fragment and GST-tagged myosin VI tail. [³⁵S]-labelled in-vitro-translated LMTK2-451-1095 was incubated with 5 µg of either GST alone (lane 2), GST-tagged myosin VI tail (lane 3) or GST-tagged myosin VI tail with the W1192L mutation (WWYWLY, lane 4). Lane 1 shows 5% of the input used for the pull down assays. Mutation of the WWY motif within the myosin VI tail dramatically reduces LMTK2 binding. (B) (C) Co-immunoprecipitation of LMTK2 and myosin VI from HeLa cells. (B) HeLa cells, untransfected (lanes 1, 2, 4) or transfected with GFP-tagged myosin VI (lane 3), were lysed and myosin VI was immunoprecipitated using polyclonal antibodies against the myosin VI tail (lanes 2-3). As a control immunoprecipitation was performed using non-immune rabbit IgG (lane 4). Lane 1 shows the whole-cell lysate, which is equivalent to 5% of the input used for each immunoprecipitation. The immunoprecipitated protein complexes were blotted with antibodies against LMTK2. Note the abnormal mobility of LMTK2 and its fragments on SDS-PAGE as described previously (Kawa et al., 2004). (C) Endogenous LMTK2 was immunoprecipitated from HeLa cells transfected with GFP–myosin-VI, and the immunoprecipitated complexes were blotted with anti-myosin VI tail antibodies. Lane 1 shows 5% of the whole-cell lysate used for each immunoprecipitation; lane 3 shows the negative control immunoprecipitation with non-immune rabbit IgG.