Plasmid Construction and Cell Transfection

Simian kidney COS-7 cells (ATCC CRL-1651) were grown at 37° C, 5% CO₂ in DMEM containing high glucose supplemented with 5% heat-inactivated fetal bovine serum (FBS), 1 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were transfected using GenJet reagent (SignaGen) according to the manufacturer instructions, and processed for analysis 48 h after transfection. The pRK5 PTEN, pRK5 GST-PTEN (N-terminal tagging), pRK5 PTEN-L (Met), and pSG5-AKT1 plasmids have been described [38 (link), 39 (link)]. pRK5 PTEN-L (Leu), pRK5 PTEN-M (Met), pRK5 PTEN-M (Ile), pRK5 PTEN-O (Met), and pRK5 PTEN-O (Leu) were made by PCR oligonucleotide site-directed mutagenesis from pRK5 PTEN-L (Met), as described [38 (link)]. Plasmid pRK5 PTEN-Δ (residues 1-343-Ser) was made by PCR mutagenesis from pRK5 PTEN. The PTEN and GST-PTEN amino acid substitution and truncation variants were made by PCR mutagenesis, and mutations were confirmed by DNA sequencing. Nucleotide and amino acid numbering for PTEN variants correspond to reference sequences from accession numbers NM_000314 and NP_000305, respectively.