Perm wash buffer

Intracellular levels of Nedd4 were measured by cytometry in rhesus macaque PBMC. Briefly, cells were pelleted by centrifugation at 1100×g for 5 min and were then resuspended in 100 µl FACS wash buffer (BD Biosciences) and stained with a cocktail of fluorochrome conjugated anti-CD3, anti-CD4 and anti-CD8 antibodies (BD Biosciences) at room temperature for 30 min. Cells were then washed with FACS wash buffer and resuspended in 250 µl Cytofix/Cytoperm buffer (BD Biosciences) for 15 min at 4°C. Following incubation, 2 ml of Perm/Wash buffer (BD Biosciences) was added to cells. Cells were pelleted by centrifugation and then resuspended in Perm/Wash buffer containing a rabbit anti-Nedd4 antibody (1∶100; Upstate, Charlottesville, VA). Cells were incubated for 15 min at 4°C, washed with 2 ml of Perm/Wash buffer followed by centrifugation and staining with a secondary goat anti-rabbit-RPE antibody (1∶50; Southern Biotech, Birmingham, AL) for 15 min at 4°C. Cells were washed with 2 ml of Perm/Wash buffer, centrifuged and then resuspended in 200 µl of FACS wash buffer (BD Biosciences). Acquisition of cells was performed by cytometry using a FACScalibur™ (BD Biosciences).