(A) CD4+ T cells were activated in vitro for 28h, treated for 1h with vehicle or a pharmacologic inhibitor of PKCζ, and stained for the proteasomal 20S α1 subunit (green), β-tubulin (red), and DNA (blue). Asymmetry of proteasome localization was observed in 61% (n=25) of vehicle-treated vs. 27% (n=29) of PKCζ inhibitor treated cells (p<0.001). (B) CD4+ T cells were activated in vitro for 48h, and control or PKCζ siRNA was introduced using electroporation. Lysates were prepared 72h later, and PKCζ and β-actin levels assessed by immunoblotting. (C) CD4+ T cells were transfected with control or PKCζ siRNA as in (B). 48h later, cells were restimulated in vitro for 24h and stained with the proteasomal 20S α1 subunit (green), β-tubulin (red), and DNA (blue). Asymmetry of proteasome localization was observed in 63% (n=60) of control transfected vs. 32% (n=62) of PKCζ siRNA transfected cells (p<0.001). Results are representative of two separate experiments.