Validating Epitope-Tagged Constructs in DU145 Cells

To positively ascertain the transfection of α5.pcDNA3.1/V5 and α5D398N.pcDNA3.1/V5 epitope-tagged constructs (a generous gift from Dr. Larry S. Barak) [40 (link)] into DU145 cells, specific immunofluorescence staining was performed on fixed cells. In brief, 24 h after nucleofection, the cells were fixed using 4% PFA in PBS (ThermoFisher, Waltham, MA, USA) for 15 min, and subsequently incubated with anti-V5 antibody (1:500, Cell Signaling, Danvers, MA, USA) overnight at 4 °C. Afterwards, cells were incubated with AlexaFluor 488 (1:1000, Invitrogen, Waltham, MA, USA) for 1 h. Samples were visualized using a Nikon Eclipse Ti2-E Widefield microscope with a Photometrics BSI camera and SpectraX laser as light source, with either a 40× or 100× objective lens.