FIGURE 1.

TβRIII suppresses Wnt/β-catenin activity at the level of signal reception. A, TβRIII mRNA expression by qRT-PCR analysis to detect endogenous TβRIII levels in OVCA429 and SKOV3 cells (i), overexpression of TβRIII in OVCA429 cells as indicated (ii), and expression in SKOV3 cells of the indicated shRNA and rescue conditions generated as described under “Experimental Procedures” (iii). Ct values are normalized in graph i to endogenous TβRIII levels in SKOV3 cells (lane 2), in graph ii to GFP (lane 1), and in graph iii to Scrambled (Scr) TβRIII levels (lane 1). Quantitations represent the average of two independent biological trials, each conducted in triplicate. B, OVCA429 cells transiently expressing increasing doses of TβRIII (5 and 10 multiplicities of infection (MOI) of TβRIII-expressing adenoviral constructs) or control (GFP) were stimulated with 50 ng ml⁻¹ Wnt3a for 1 h followed by immunoblotting of lysates for phospho-LRP6 (Ser-1490) (pLRP6), LRP6, TβRIII, and β-actin. C, SKOV3 cells transiently expressing shRNA to TβRIII (shTβRIII-1) or Scrambled control (“Experimental Procedures”) and transiently transfected with rat TβRIII (rTβRIII) or control vector (pcDNA 3.1) for 24 h for rescue of TβRIII expression (as seen in A, right panel) and then stimulated with 50 ng ml⁻¹ Wnt3a for 1 h followed by immunoblotting of lysates for phospho-LRP6 (Ser-1490) (pLRP6), LRP6, and β-actin. D, SKOV3 cells transiently expressing a second independent shRNA to TβRIII (shTβRIII) or Scrambled control (“Experimental Procedures”) were stimulated with 50 ng ml⁻¹ Wnt3a for 1 h followed by immunoblotting of lysates for phospho-LRP6 (Ser-1490) (pLRP6), LRP6, and β-actin. B–D, quantitations represent pLRP6:LRP6 ratios and are normalized to the untreated sample. All panels (A–D) represent at least two independent biological trials.