Figure 3.

Identification of Bcl-G_L as an interacting protein for MELK. (a) Silver staining of SDS-PAGE gels that contained the pulled-down cell lysates. The enlarged area covering Bcl-G shows the differential interaction between it and wild-type MELK (WT-MELK) and kinase-dead MELK (D150A-MELK). (b) Expression of Bcl-G_L in eight breast cancer cell lines as well as human mammary epithelial cells (HMECs) by western blot analysis with an anti-Bcl-G_L antibody. β-Actin was used as a control. (c) Interaction of MELK with Bcl-G_L. Extracts from HeLa-cells transfected with HA (hemagglutinin)-tagged WT-MELK (HA-WT-MELK) or Flag-tagged Bcl-G_L (Flag-Bcl-G_L), or a combination of these, were harvested 36 hours after transfection. The cell lysates were immunoprecipitated with anti-Flag M2 antibody. Precipitated proteins were separated by SDS-PAGE and western blotting analysis was performed with an anti-HA antibody. (d) Direct interaction of the MELK and Bcl-G_L proteins. The upper panel indicates the amount of input of WT-MELK and D150A-MELK. His-tagged WT-MELK bound to Bcl-G_L, but His-tagged D150A-MELK did not. (e) Schematic representation of the amino- and carboxy-terminal deletion constructs of Bcl-G_L. The C-1 and C-2 constructs have the BH2 domain deleted, and the C-3 construct has both the BH2 and BH3 domains deleted. (f) Determination of the WT-MELK binding regions of Bcl-G_L by immunoprecipitation. The HA-tagged WT-MELK and various peptide sequences of Flag-tagged Bcl-G_L (Figure 3e) were pulled down by immunoprecipitation with Flag-M2 antibody and then immunoblotted with rabbit anti-Flag antibody. The expression of HA-tagged WT-MELK in total cell lysates was confirmed by western blotting analysis. As a control, immunoprecipitation was performed from cells co-transfected with pCAGGSn3FC (Mock) and HA-tagged WT-MELK (HA-WT-MELK) through all steps. Arrowheads indicate expression of each Bcl-G_L peptide.