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. 2021 May 28;15(1):148. doi: 10.3892/mco.2021.2310

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Copyright: © Kitazawa et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Sequence analysis of genes inserted into the pMXs-IRES-GFP vector. Flow cytometery and western blotting demonstrated successful ectopic KRAS gene transduction. (A) The KRAS mutant genes inserted into the vector were amplified using the BigDye™ Terminator v3.1 Cycle Sequencing kit with a vector primer (5'-GACGGCATCGCAGCTTGGATACAC-3') and analyzed using an ABI 3130 Genetic Analyzer. (B) Flow cytometery analysis of GFP-positive rates in a mixed state with GFP-positive transgenic cells (mock, KRAS wild, G12V and G12C) and GFP-negative parental cells. (C) Transduction of KRAS wild and mutant (G12V and G12C) genes were confirmed via western blotting using a FLAG-specific antibody. MEK and ERK phosphorylation in CACO-2 cells was also examined via western blotting. (D) Normalized band intensity of FLAG relative to actin, pMEK relative to MEK and pERK relative to ERK. ^*P<0.05 vs. KRAS-wild type. KRAS, Kirsten rat sarcoma 2 viral oncogene homolog; GFP, green fluorescent protein; p, phosphorylated.