Transfection with PTPε siRNA and pMXs-STAT3C

The Neon™ Transfection System (Invitrogen, Carlsbad, CA) was used for transfection via electroporation. Transfection efficiency was measured using Western blot analysis. MEF cells were resuspended in 120 μL of Neon Resuspension Buffer R for every one million cells in preparation for transfection. For each electroporation, DU145 cells and 10 μL of PTPε siRNA (50 nM, Santa Cruz Biotechnology, Santa Cruz, CA) were aliquoted into a sterile microcentrifuge tube. MEF cells and 1 μg of pMXs and pMXs-STAT3C plasmids were aliquoted into a sterile microcentrifuge tube. A Neon Tip was inserted into the Neon Pipette and the cell-plasmids mixture was aspirated into the tip while avoiding the formation of air bubbles. The Neon Pipette was then inserted into the Neon Tube containing 3 mL of Neon Electrolytic Buffer E in the Neon Pipette Station. DU145 cells were subjected to a single 1,260-volt pulse with a width of 20. MEF cells were subjected to a single 1,350-volt pulse with a width of 30. 48 h after transfection, cells were treated with 5 μM Capz for 6 or 24 h, and whole-cell extracts were prepared for measurement of PTPε, p-STAT3(Tyr705), PARP, and β-actin by Western blot.