Figure 4

Activation of Wnt/β -catenin signaling pathway by CDKAL1. (a) Western blotting of β-catenin in 3T3-L1 expressing CDKAL1 on day 0 and day 6 of differentiation. β-actin was used as an internal control for protein loading. The experiments were repeated multiple times and representative blot was shown. (b) Western blotting of active (unphosphorylated) β-catenin in the nuclear fraction in 3T3-L1 expressing CDKAL1. (c,d) The effect of stable knockdown of CDKAL1 by shRNA in 3T3-L1 cells on protein levels of active β-catenin in total lysate (c) and in the nuclear fraction (d). The inset graph in (d) shows quantification of the active β-catenin (*p < 0.05 vs control group by Student’s t-test). (e) Western blotting of β-catenin and inactive form of GSK-3β (phosphorylated at Ser 9) in 3T3-L1 expressing CDKAL1. (f) Measurement of Wnt signaling activities by TOPFLASH reporter assay in 3T3-L1 cells. FOPFLASH reporter was used as the negative control (n = 6, *p < 0.05 vs control group by Student’s t-test). (g) Gene expression changes of Wnt target genes in 3T3-L1 cells expressing CDKAL1 (12 hours after the initiation of differentiation, n = 3, ***p < 0.001 vs control group by Student’s t-test).