FIGURE 2.

Endogenous GSKIP enhances Wnt signaling through controlling PKA phosphorylation of β-catenin at Ser-675. HEK293, A549, and HeLa cells were left untreated or treated as indicated with siRNA to knock down GSKIP (siGSKIP), control siRNA (siNT), Wnt3a-enriched medium, or control medium in the absence or presence of the PKA inhibitors, PKA inhibitor peptide (PKI) or H89, or the proteasome inhibitor MG-132. A–C, cytosol fractions were purified from the three cell types and analyzed by Western blotting (WB) for expression of the indicated proteins. The signals were densitometrically analyzed, and ratios of β-catenin/GAPDH and Ser(p)675-β-catenin/GAPDH were calculated; n ≥ 6, mean ± S.E., ANOVA, **, p < 0.01, ***, p < 0.001. D, TOPflash/FOPflash (TOP/FOP) luciferase assay. HEK293 cells were stimulated with Wnt-enriched or control medium; n = 4, mean ± S.E., ANOVA, **, p < 0.01. E, cell lysates were purified from HEK293 cells, and the indicated proteins were detected by Western blotting. Semi-quantitative densitometric analysis of Western blots is depicted as ratios of Ser(P)1490 LRP6/GAPDH and Ser(P)9 GSK3β/GAPDH; n = 6, mean ± S.E., ANOVA, *, p < 0.05, ***, p < 0.001. F, cytosolic fractions were purified, and the indicated proteins were detected by Western blotting. Semi-quantitative densitometric analysis of Western blots shows the ratios of β-catenin/GAPDH and (Ser(P)33/Ser-37/Thr-41)-β-catenin/GAPDH; n = 6, mean ± S.E., ANOVA, ***, p < 0.001. pS33/S37/T41, Ser(P)33/Ser-37/Thr-41. G, the enrichment of the indicated proteins in the cytosolic fractions of HEK293 cells that were used in A was confirmed by Western blotting. The cells were treated with Wnt3a-enriched or control medium. Shown are representative blots from n ≥ 4 experiments.