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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Cell Signal. 2015 Feb 12;27(5):961–968. doi: 10.1016/j.cellsig.2015.02.005

Figure 2. LPA increases phosphorylation of β-catenin at Ser552 and Ser675.

Figure 2

(A) HCT116 cells transduced with shCont or shLPA2, and LPA2 knockdown efficiency was evaluated by qRT-PCR. (B) HCT116 cells transduced with shCont or shLPA2 were treated with LPA for 15 min, and phosphorylation of GSK-3β (Ser9) and β-catenin (Ser552 and Ser675) was determined. β-actin was used as a loading control. All figures are representatives of at least three experiments. Relative phosphorylation levels were quantified by densitometric analysis and expressed as percent changes relative to cells without LPA under each condition. (C) Cells were pretreated with H89 (10 µM) or LY294002 (10 µM) for 30 min prior to stimulation by LPA for 15 min. Phosphorylation of d β-catenin was determined as described above. Protein band intensity is expressed as a percent change relative to control without LPA. n=3. (D) Expression levels of p-S552 and p-S675 β-catenin were determined in cytoplasmic and nuclear fractions. β-tubulin and Lamin B1 were used as cytoplasmic and nuclei loading control, respectively. (E) Cells transiently transfected with FLAG-tagged WT, S552A, S675A, or S552A/S675A (AA) β-catenin were treated with LPA for 4 h. The β-catenin/TCF transcriptional activity was assayed using TOPflash as described above. Data (means ± SE) presented are the relative luciferase activity of three independent experiments. *, p < 0.01. **, p < 0.05. n=3.