2.7. Transfection of Cells

As published previously [14], the zyxin expression plasmid was constructed by subcloning a full-length polymerase chain reaction fragment (PCR) including the first stop codon (position 305 to 1999; NM 011777) derived from VSMC cDNA into the cDNA 6.2/N-EmGFP TOPO 5.9-kb vector using the TOPO cloning reaction according to the manufacturer’s instructions (TOPO Mammalian Expression Vector Kit, Invitrogen). The same principle was applied for the LPP expression plasmid that encompassed the full-length PCR fragment with the first stop codon (position 551 to 2392, NM 178665.5). A GFP-expressing construct (Lonza Bioscience via Biozym Scientific, Hessisch Oldendorf, Germany) was used as a control for all transient transfection experiments. Nucleofector^TM technology (Lonza Bioscience) was used for transient transfection of the expression plasmids into the cultured VSMCs according to the manufacturer’s instructions. The transgenic expression of LPP and zyxin was confirmed by immunofluorescence and Western blot analyses. Cells were analysed 48 h post transfection.