Figure 6

LKB1 interacts with endogenous Dbl. A. Schematic representation of proto-Dbl and the constructs used in this work. B. HeLa-S3 cells transfected with vector (pRK5myc), or pRK5myc-wild type LKB1. LKB1 was immunoprecipitated from cell lysates with anti-myc (lanes 1 and 3), or control IgG (lane 2) antibody and analyzed by western blot analysis. Dbl was detected using a Dbl antibody. Approximately 20% of endogenous Dbl was precipitated with myc antibody. C. HeLa-S3 cells transfected with pRK5myc-LKB1. Cell lysates were subjected to immunoprecipitation using either control rabbit IgG (lane 1), or anti-Dbl antibody (lane 2). Upper two panels are total cell lysates blotted with anti-Dbl and anti-myc antibodies. Lower two panels are Dbl and IgG immunoprecipitates blotted with anti-Dbl and anti-myc antibodies. Around 1% of the overexpressed myc-LKB1 was precipitated with endogenous Dbl using anti-Dbl. D. HeLa-S3 cells transfected with pRK5myc, or pRK5myc with LKB1 cDNAs. Bottom two panels: Endogenous Dbl immunoprecipitated from cells lysates with anti-Dbl antibody, followed by western blot using either an anti-myc, or anti-Dbl antibody. Top panel: Expression of LKB1 in total cell lysates using anti-myc. E. Dbl constructs expressed in HEK293T cells as GST fusion proteins and purified using glutathione agarose. Equal amounts of cell lysate isolated from cells expressing pRK5myc-wild type LKB1 were added to the beads, and bead-associated LKB1 detected on western blot analysis using anti-myc antibody (bottom panel). GST-Dbl and LKB1 were detected with anti-GST and anti-myc antibody, respectively.