Figure 3

Tiam1 lies upstream of Rac1-mediated activation of the canonical Wnt pathway. (A) HCT116 cells were co-transfected with pTOPFlash or pFOPFlash reporter plasmids and pCMVβ-galactosidase, in addition to the indicated expression plasmids: wild-type (wt) Rac1 alone (0.4 μg), Tiam1 alone (0.5 μg), wt Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively), and N17Rac1 and Tiam1 in combination (0.4 μg and 0.5 μg, respectively). Empty vector was transfected to establish basal TOPFlash activity. Luciferase activity was normalized to β-galactosidase expression, and is expressed as total relative light units (RLU). Results shown are representative for three independent experiments performed in triplicate. Bars represent mean ± S.E. and *p < 0.05. (B) HCT116 cells were transiently co-transfected with either Tiam1-specific siRNA or control, and with either pTOPFlash or pFOPFlash reporter plasmids for 64 hours. Total RNA was analyzed by real time RT-PCR for the detection of Tiam1 transcript levels, which were normalized to transcript levels of β-actin (left). Luciferase activity was measured and normalized to β-galactosidase expression, and is expressed as total relative light units (right). Bars represent mean ± S.D. and *p < 0.05. (C) HCT116 cells were transfected with indicated plasmids and immunoprecipitated (IP) with β-catenin antibody. Immunoblotting (IB) was performed using β-catenin and myc-tag antibodies to detect myc-tagged wild-type (wt) Rac1. Whole cell lysates (WCL) were immunoblotted with β-catenin, myc-tag, or HA-tag antibodies to confirm expression of β-catenin, wild-type Rac1, or Tiam1, respectively. (D) Whole cell extracts of HCT116 cells were immunoprecipitated (IP) with Tiam1 antibody and analyzed by Western blotting (IB) using Tiam1, β-catenin, or Rac1 antibodies. Endogenous expression of these proteins was confirmed by immunoblotting whole cell lysates with indicated antibodies.