Fig. 3

Figure 3. Overexpression or knockdown of VBP1 promotes protein degradation of TCF/LEFs. A, exogenous Tcf/Lef protein levels in control or VBP1-overexpressing HEK293T cells. Similar results were obtained from four experiments. B, endogenous TCF7L2 protein level in control or VBP1-overexpressing HEK293T cells. Similar results were obtained from three experiments. C, changes in exogenous TCF7L2 protein level in VBP1-overexpressing HEK293T cells treated with MG132. Cells transfected with indicated plasmid DNA and treated with or without 10 µm MG132 for 8 h. Similar results were obtained from three experiments. D, TCF7L1 and TCF7L2 protein levels in control or VBP1-knockdown HEK293T cells. Similar results were obtained from three experiments. E, qRT-PCR analysis of transcriptional levels of TCF7L1 and TCF7L2 in control and VBP1-knockdown HEK293T cells. Data are from three independent experiments with individual data points shown. F, representative image of TCF7L2 protein levels in control or VBP1-knockdown HEK293T cells treated with CHX in a time series. G, quantitation of relative protein levels from the two isoforms of TCF7L2, TCF7L2E (left panel) and TCF7L2M/S (right panel), in (F), respectively. The band intensity was quantified using ImageJ software. Data are from three independent experiments. H, TCF7L1 and TCF7L2 protein levels in control or VBP1-knockdown HCT116 cells. Similar results were obtained from three experiments. I, changes in TCF7L2 protein levels in VBP1-knockdown HEK293T cells treated with MG132. VBP1-depleted HEK293T cells treated with or without 10 µm MG132 for 8 h. Similar results were obtained from three experiments. J, changes in TCF7L2 protein levels in VBP1-knockdown HCT116 cells treated with MG132. VBP1-knockdown HCT116 cells treated with or without 10 µm MG132 for 8 h. Similar results were obtained from three experiments. K, ubiquitylation assays in HEK293T cells transfected with indicated plasmids. HIF-1α was used as a positive control of ubiquitylation promoted by VBP1. Similar results were obtained from three experiments. The values are mean ± S.D. Unpaired t test. *p < 0.05; **p < 0.01; ***p < 0.001; ns, nonsignificant.