Pcdna3 ha vector

To construct pcDNA3-HA-RasG12V, an expression vector that contain full-length human G12V-H-Ras, the human RasG12V cDNA was amplified with a pair of primers (a forward primer: 5′-GCGAATTCATGACGGAATATAAGCTGGTG-3′ and a reverse primer: 5′-GCAGCGGCCGCTCAGGAGAGCACACACTTG-3′) using a vector, pCMV-HA-RasG12V (kindly provided by Dr. K Kaibuchi, Nagoya University, Japan), as a template. The resulting fragment was digested with EcoRI and NotI and cloned into downstream of the HA tag sequence in the pcDNA3-HA vector (Invitrogen). For construction of pcDNA3-HA-DAO, an expression vector of HA-tagged human full-length DAO (NCBI accession number: NM_001917.4), the DAO cDNA was amplified with a pair of primers (a forward primer: 5′-CGTGCTCGGAATTCATGCGTGTGGTGGTGATTGGAG-3′ and a reverse primer: 5′-CGTGCTCGGCGGCCGCTCAGAGGTGGGATGGTGGCATT-3′) using a cDNA sample prepared from U2OS cells as a template, and the resulting fragments were digested with Eco RI and Not I, and cloned into downstream of the HA tag sequence in the pcDNA3-HA vector. To generate a catalytically inactive DAO mutant (R199W), PCR reactions were performed using mutagenic primers (a forward primer: 5′-CCCCTGCTGCAGCCAGGCTGGGGGCAGATCATGAAGG-3′ and a reverse primer: 5′-CCTTCATGATCTGCCCCCAGCCTGGCTGCAGCAGGGG-3′).