Silencing and Tagging of Vinculin

Endogenous vinculin was silenced using pSUPER-shVIN that was generated by subcloning an oligo targeting the human vinculin sequence TTCAAGAGA into the retroviral vector pSUPER-RETRO-PURO (Oligoengine; Peng et al., 2010 (link)). pLEGFP-WT vinculin was generated by amplifying full-length chicken vinculin, ligating it into the pENTR-DTOPO and then cloning it into a pLEGFP-DEST vector using the Gateway cloning system (Invitrogen). pLEGFP-DEST was digested with HindIII and BamH1 and ligated into pLEGFP-C1 vector and expression driven by CMV promoter. pLEGFP-vinculin Y822F was prepared using site-specific mutagenesis to introduce the appropriate single amino acid substitution into pLEGFP-WT vinculin (Peng et al., 2010 (link)). Full-length Snail was cloned into the pQCXIP retroviral vector and expression driven by CMV promoter (Place et al., 2013 (link)). pGEX4T1-vinculin 811–881 were constructed by PCR amplifying corresponding residues of chick vinculin and subcloning this into pGEX4T1 (GE Healthcare). pGEX4T1-vinculin 811–881 Y822F were prepared by using site-specific mutagenesis to introduce a mutation resulting in the appropriate single amino acid substitution into pGEX4T1-vinculin 811–881.