Ferlito et al. 10.1073/pnas.0600275103. |
Supporting Materials and Methods
Supporting Materials and Methods
Cell Culture. LAK cells were prepared from the blood of healthy human volunteers in a protocol approved by The Johns Hopkins University School of Medicine Institutional Review Board (1). LAK cells were transfected by using an electroporator (Nucleofector, Amaxa, Gaithersburg, MD). To explore the role of Ras in mediating NO inhibition of killing, we transfected LAK cells with the vector dsRED, expressing a red fluorescent protein (Clontech, Mountain View, CA), a vector expressing dsRED-N-Ras (a generous gift from Philippe Bastiaens, EMBL Heidelberg, Heidelberg, Germany), or a vector expressing GFP-Rac-1 (a generous gift from Subhendra Mattagajasingh, University of Pittsburgh, Pittsburgh, PA) (2).
Assay of LAK Cell Killing of K562 Cells.
To measure LAK cell killing of K562 cells, cells were plated at a 1:3 ratio of target K562 cells to LAK effector cells (1 × 105 K562 cells/3 × 105 LAK cells) for 0-3 h in round-bottom 96-well plates (1, 3). In some experiments, DETA-NONOate or control was first added to LAK cells for 0-18 h, and the LAK cells were washed and cocultured with K562 cells. In other experiments, RAW cells were treated with LPS and IFN-g for 18 h to activate iNOS expression and washed, and then LAK cells were plated onto permeable transwell inserts adjacent to and above the stimulated RAW cells for 6 h. LAK cells were then removed, washed, and cocultured with K562 cells.Immunoprecipitation and Immunoblotting.
Immunoprecipitation and immunoblotting was performed as described in ref. 4.1. Andrade, F., Roy, S., Nicholson, D., Thornberry, N., Rosen, A. & Casciola-Rosen, L. (1998) Immunity 8, 451-460.
2. Rocks, O., Peyker, A., Kahms, M., Verveer, P. J., Koerner, C., Lumbierres, M., Kuhlmann, J., Waldmann, H., Wittinghofer, A. & Bastiaens, P. I. (2005) Science 307, 1746-1752.
3. Topalian, S. L., Solomon, D. & Rosenberg, S. A. (1989) J. Immunol. 142, 3714-3725.
4. Matsushita, K., Morrell, C. N., Cambien, B., Yang, S. X., Yamakuchi, M., Bao, C., Hara, M. R., Quick, R. A., Cao, W., O’Rourke, B., et al. (2003) Cell 115, 139-150.