Fig. 6. Plk1 regulates the association of Nlp with the dynein-dynactin complex. (A) U2OS cells were transfected with EGFP-Nlp or EGFP-Nlp8 (green) together with either Plk1^K82R or Plk1^T210D, and the localisation of p150^Glued (red) was analysed by immunofluorescence microscopy. Bar, 10 µm. (B) Nlp was immune precipitated from extracts of `Tet-On' Myc-Nlp-expressing cells using antibodies to the Myc-epitope; control precipitations were performed using a non-specific IgG fraction. Samples of the cell lysate, unbound, and bound fractions were western blotted for Nlp and p150^glued. (C) Total cell extracts were incubated with the GST-Nlp N terminus, GST-Nlp N terminus mock-treated with Plk1 and GST-Nlp N terminus phosphorylated by Plk1. Bound fractions were analysed by immunoblotting using antibodies to p150^Glued and Plk1 (left panel), and Coomassie Brilliant Blue staining (right panel).