Pcdna3 s33y β catenin

Mouse Axin2 cDNA was made by RT-PCR of total RNA isolated from mouse vagina tissue and was cloned into pIVT vector (Igarashi et al., 2007 (link)) between the SalI and XbaI sites. Axin2V26D mutant construct was made using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA). pcDNA3-S33Y β-catenin was purchased from Addgene (Kolligs et al., 1999 (link)). A FLAG tag was added to the C-terminus, a 2X nuclear localization signal was added to the N-terminus, an AgeI restriction site was added to the 5’ end, and an XbaI restriction site was added to the 3’ end of β-catenin-S33Y via PCR. pGEMHE-mCherry-Trim21 purchased from Addgene (Clift et al., 2017 (link)) was digested with AgeI and XbaI to remove the mCherry-Trim21 sequences and β-catenin-S33Y was cloned into the pGEMHE vector between the AgeI and XbaI sites. pFLAG-TNKS-2 was purchased from Addgene (Sbodio et al., 2002 (link)). An AgeI restriction site was added to the 5’ and an Xba1 site to the 3’ end of TNKS-2 via PCR. TNKS-2 was cloned into the pGEMHE vector between the AgeI and XbaI sites. For microinjection, cRNAs were made by in vitro transcription using the AmpliCap-Max T7 High Yield Message Maker Kit (CELLSCRIPT, Madison, WI).