Figure 6

Treatment with proteasome inhibitor, Lactacystin, fails to affect constitutive levels of IκBα in human T cells. T cells obtained from young and elderly donors were either left untreated (-) or treated with lactacystin (5 μM) for 2 h to inhibit the proteasome. At the end of incubation cells were washed and lysates were prepared as described. Equal amounts of protein from cell lysates were resolved using SDS-PAGE and electroblotted. Resolved IκBα was detected using antibody to IκBα and ECL. To ensure equal protein loading, blots were stripped and reprobed with β-actin. Representative data from one donor pair are presented (A). As a positive control for lactacystin, T cells from young and elderly donors were either pretreated with Lactacystin (5 μM, 2 h) and then activated with TNFα for 10 min (L+T) or activated with TNFα without lactacystin pretreatment (T). US represent untreated T cells. Cell lysates were resolved using SDS-PAGE and IκBα detected as indicated above (B).