Fig 6. Proper expression of p120ctn and E-cadherin is required for endoderm differentiation from mESCs.

(A) Immunoprecipitation (IP) experiments for p120ctn-null mESCs with R26-based expression of p120ctn isoform 1A (R1A WT) or its corresponding mutant (R1A K401M). (B) IP experiments for p120ctn-null mESCs with R26-based expression of p120ctn isoform 3A (R3A WT) or its corresponding mutant (R3A K401M). IPs were performed with an anti-p120ctn antibody (IP_Rel) or with an irrelevant anti-GFP antibody (IP_Irrel). Eluates immunoblotted (IB) with an anti-p120ctn antibody (top panels) showed that p120ctn was efficiently bound to the beads. Immunoblotting with an anti-E-cadherin antibody (bottom panels) confirmed the interaction of wild-type p120ctn with E-cadherin, whereas mutated K401M p120ctn was unable to bind E-cadherin. (C) Immunohistochemistry for p120ctn, E-cadherin and AFP on DIV30 p120ctn^-/- EBs expressing from the endogenous R26 promoter either p120ctn isoform 3A (R_p120_3A) or its E-cadherin-uncoupled K401M mutant form (R_p120_3A_K401M). Scale bars: 50 μm. (D) qRT-PCR analysis for endoderm-specific marker genes was performed using cDNAs originating from DIV12 control and p120ctn^-/-;AL^tg/+ EBs, and from p120ctn^-/-;AL^tg/+ EBs expressing various rescue constructs from the R26 promoter as indicated by the EB names. Tbp and Rpl13a were used for normalization. The error bars in the graphs represent the standard deviation of three technical replicates. * or ** denote comparisons that are significantly different. The P values (t test) from left to right are as follows: 0.016, 0.014 and 0.0018.