Skip to main content
. 2012 Dec 31;288(6):3753–3767. doi: 10.1074/jbc.M112.415240

FIGURE 7.

FIGURE 7.

A, depletion of HectD1 augmented Wnt3a-induced β-catenin stabilization and transcription. HEK293 cells were co-transfected with the indicated siRNAs and luciferase reporter plasmids (pTOP-Flash and pCMV-Renilla) for 48 h. The cells were then treated with vehicle (PBS + 0.2% BSA, −) or Wnt3a (100 ng/ml) for 24 h. Equal amounts of lysates were analyzed by immunoblotting with the indicated antibodies or assayed for TOP-Flash luciferase reporter activity (bottom panel). Knockdown of β-catenin served as a negative control. The anti-CtBP1 blot indicates loading equivalence. The error bars indicate standard deviation of triplicate samples. The data shown are representative of three independent experiments. B, depletion of Trabid suppresses β-catenin-mediated transcription. HEK293 cells were co-transfected with the indicated ON-TARGETplus-modified Trabid-specific siRNAs and TOP-Flash luciferase reporter plasmids as in A. The cells were treated with Wnt3a and processed for immunoblotting or TOP-Flash assays as described in A. After 72 h of transfection, all Trabid-specific siRNAs reduced ZRANB1 transcript levels by at least 90% (quantified by quantitative RT-PCR; data not shown). The anti-β-actin blot indicates loading equivalence. The error bars indicate standard deviation of triplicate samples. The data shown are representative of two independent experiments. C, HEK293 cells were co-transfected with two additional ON-TARGETplus-modified HectD1-specific siRNA oligonucleotides (#3 and #4) and TOP-Flash luciferase reporter plasmids, followed by Wnt3a treatment as described for A. The error bars indicate standard deviation of triplicate samples. The data shown are representative of three independent experiments. D, RPE cells were transfected with control or HectD1-specific siRNA 3 for 48 h and then treated with vehicle (−) or Wnt3a (100 ng/ml) for 24 h. The cells were harvested and divided into two equal portions to prepare lysates for immunoblotting with the indicated antibodies or processed for quantitative RT-PCR to quantify the expression of AXIN2 (lower panel). The error bars indicate standard deviation of triplicate samples. The data shown are representative of three independent experiments. E, HEK293 cells were co-transfected with the indicated siRNAs and TOP-Flash luciferase reporter plasmids, followed by Wnt3a treatment as described for A. The cells were harvested, and the lysates were used for TOP-Flash assays as described for A. The error bars indicate standard deviation of triplicate samples. The data shown are representative of two independent experiments.