MKlp2 is a novel binding partner of myosin-II <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>A</b>) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. (<b>B</b>–<b>D</b>) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. (<b>E</b>) Autoradiography of <i>in vitro</i>-translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total <i>in vitro</i>-translated product is shown. (<b>F</b>) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with <i>in vitro</i>-polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.