Cloning and Mutagenesis of Tgm2 in pcDNA3

The pcDNA3 and pcDNA3-Fyn-CA-V5 plasmids were constructed as previously published [16 (link)]. The IMAGE clone 3256943 (GenBank: BC016492) for mouse Tgm2 was obtained from imaGenes (Berlin, Germany) and PCR was performed with a pair of oligonucleotides (5′-CACCATGGACTACAAGGACGATGACGACAAG-ATGGCAGAGGAGCTG-3′ and 5′-TTAGGCCGGGCCGATGATAA-3′). The PCR product was separated on a 2% agarose gel and the specific single band was extracted using the QIAquick PCR purification kit (Qiagen, Venlo, The Netherlands). The purified PCR product was cloned into pcDNA3.1D/V5-His-TOPO using the pcDNA3.1 Directional TOPO Expression Kit (ThermoFisher). The pcDNA3.1-Flag-Tgm2-Y369 construct was obtained using the QuickChange 2-XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) with a pair of oligonucleotides (5′-GAAGAGCGAAGGGACATTCTGTTGTGGCCCA-3′ and 5′-TGGGCCACAACAGAATGTCCCTTCGCTCTTC-3′).