Nak1 and Orb6 interact <i>in vitro</i> and <i>in vivo</i>. A.

Abstract

<p>Extracts from WT (SP199) cells expressing HA-Orb6 and control vector (lane 1), Myc-Nak1 and control vector (lane 2), or HA-Orb6 and Myc-Nak1 (lane 3) from <i>nmt1</i> promoter expression plasmids were analyzed by western-blots using anti-Myc (9E10) or anti-HA (12CA5) monoclonal antibodies (Input panels). HA-Orb6 was immunoprecipitated from cell extracts with anti-HA antibody and equal portions of the immunoprecipitates were probed with anti-HA antibody and anti-Myc antibody (αHA IP panels). <b>B.</b> Interaction of purified recombinant GST-Orb6 and His₆-HA-Nak1 was assayed by an <i>in vitro</i> binding assay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037221#s4" target="_blank">Materials and Methods</a>). The left panel shows Coomassie blue staining of purified recombinant GST (lane 1), GST-Orb6 (lane 2), His₆-vector control (lane 3), His₆-HA-Nak1 (lane 4). Purified His₆-HA-Nak1 (input, lane 7) was incubated with either GST (lane 5) or GST-Orb6 (lane 6) bound to Glutathione Sepharose 4B beads. The right panel shows a Western blot using anti-HA (12CA5) antibody of His₆-HA-Nak1 bound to the beads. <b>C.</b> Schematic diagram of mutant Nak1 expression constructs. The Nak1 N-terminal kinase domain (residues 1–262) and C-terminal (CTR) region (554–652) are indicated. The numbers at the left and the bars at the right indicate the region of Nak1 encoded by the various deletion constructs. <b>D.</b> Extracts from wild-type (SP199) cells co-expressing Myc-Orb6 with the control vector (lane 1), HA-Nak1^1–562 (lane 2), HA-Nak1^1–585 (lane 3), HA-Nak1^1–607 (lane 4), HA-Nak1^1–629 (lane 5), HA-Nak1 (lane6), or HA-Nak1^554–652 (lane 7) were analyzed by western-blots using anti-Myc (9E10) or anti-HA (12CA5) monoclonal antibodies (Input panels). Extracts were immunoprecipitated with anti-HA antibody and equal portions of the immunoprecipitates were probed with anti-HA antibody and anti-Myc antibody (αHA IP panels).</p