C terminal v5 tag

The sequences encoding the respective splice variants P2-MDM2-10 and MDM2-∆5 were assembled from synthetic oligonucleotides and cloned into E. coli expression vectors by Geneart, (Life Technologies). MDM2 encoding fragments were cut out using the BamHI and XhoI restriction sites. Following agarose gel purification, the fragments were ligated into a pCMV eukaryotic expression vector (CMV-MCS-V5-6xHis-BGHpolyA in pCMV-cyto-EGFP-myc) using T4 DNA ligase. The utilized vector contained a sequence encoding an eGFP expressed from an independent CMV promoter region. Performing immunofluorescence, apoptosis, and senescence analysis, a pcDNA3.1V5-vector (TOPO) was used, providing a C-terminal V5-tag (Invitrogen). The plasmids were amplified in One Shot TOP10 Chemically Competent E. coli cells (Invitrogen) by ampicillin selection followed by colony PCR and purified using the QIAprep Spin Miniprep Kit (Qiagen). The constructed plasmids encoding MDM2-FL and splice variants were confirmed and checked for mutations by sequencing using the BigDye1.1 system and Sanger sequencing prior to large-scale purification from E. coli by the HiSpeed plasmid maxi kit (Qiagen) according to the manufacturer's instructions. The resulting stock solutions of the plasmids were validated by sequencing to ensure absence of any mutations prior to introduction to a eukaryotic cell system.