FIGURE 1.

RACK1 attenuates Dvl-mediated Wnt signaling. A, after transfection with Topflash luciferase with or without Myc-RACK1 for 24 h, HEK293T cells were treated with control medium or Wnt3a conditioned medium (CM) overnight, and then luciferase activity was measured. The pRL-TK Renilla reporter was co-transfected to normalize transfection efficiency. Myc-RACK1 was transfected with gradients of 50 or 100 ng. B, HEK293T cells were transfected with Topflash luciferase, Myc-RACK1, and FLAG-Dvl2 as indicated for 24 h and harvested for luciferase activity determination. C, HEK293T cells were transfected with or without Myc-RACK1. After 24 h, the cells were treated with Wnt3a CM for 6 h and then harvested for immunoblotting. Tubulin served as a loading control. D, HEK293T cells were transfected with Topflash luciferase (Luc) for 24 h after being infected by a lentivirus expressing nonspecific or RACK1 shRNA. Cells were then treated with control (Con) medium or Wnt3a CM overnight, and then luciferase activity was measured. The pRL-TK Renilla reporter was co-transfected to normalize transfection efficiency. E, HEK293T cells were infected by a lentivirus expressing nonspecific or RACK1 shRNA. After 48 h, the cells were treated with Wnt3a CM for 6 h and then harvested for IB. The protein levels of β-catenin were quantified and normalized against tubulin, and the values are shown below the bands. All reporter assays were performed in triplicate, and the data are represented as mean ± S.D. after being normalized against Renilla activity. Statistical analysis was performed with two-tailed unpaired Student's t test (**, p < 0.01).