Fig. 1. Wnt signaling regulates CK1α levels via ubiquitin-dependent proteasomal degradation.

a Extracts of HEK cells treated with Wnt3a for various lengths of time were evaluated by immunoblotting. A representative immunoblot (left panel) or quantitation of immunoblots (mean ± SEM, n = 4 independent experiments; right panel) is shown. b RNA from HEK cells treated with Wnt3a for various lengths of time was used to determine the expression of the indicated genes, using quantitative RT-PCR analysis. Quantification of gene expression (mean ± SEM, n = 3 independent experiments) is shown. c HEK cells were co-treated with cycloheximide and PBS or Wnt3a for the indicated time and extracts of these cells were evaluated by immunoblotting for CK1α or HSP90. CK1α levels from immunoblots were quantitated, normalized to that of HSP90, and plotted to determine CK1α turnover in response to Wnt3a (mean ± SEM, n = 3 independent experiments). Asterisks indicate statistical significance (two-way ANOVA analysis, ****p value < 0.0001). d Extracts of HEK cells treated for 6 h with PBS or Wnt3a, together with DMSO or 10 μM MG132, were evaluated by immunoblotting. A representative immunoblot (left panel) and a quantification of immunoblots from (mean ± SEM, n = 3 independent experiments; right panel) are shown. e Extracts of HEK cells treated for 24 h with PBS or Wnt3a, together with DMSO or 20 nM bafilomycin A1 (BA), were evaluated by immunoblotting. A representative immunoblot (left panel) and a quantification of immunoblots (mean ± SEM, n = 3 independent experiments; right panel) are shown. P62 is a biomarker for lysosomal/autophagosomal inhibition. Asterisks in d and e indicate statistical significance (two-tailed Student’s t-test, *p value < 0.05, ***p value < 0.001). f CK1α was immunoprecipitated from the lysates of HEK cells treated with Wnt3a and MG132 for various time points, followed by analyses of the indicated proteins by immunoblotting. A representative immunoblot (n = 3 independent experiments) is shown.