p30 binding to PA28γ does not inhibit PA28γ proteasomal activity. (A-B) Cell lysates from cells expressing p30 (open triangles) or mock-transfected cells (closed squares) were used in proteasomal activation assays as described in “Methods.” Samples were assayed for their ability to cleave the fluorescent substrates LLVY (A) or LRR (B), which detect the activity of PA28α and PA28γ, respectively. (C) Cell lysates from panel A were Western blotted as indicated. (D) Eluted protein complexes isolated as in panel A were subjected to proteasomal activity assays as described in panel A. Mock-transfected (open circles), PA28γ (closed squares), PA28γ + p30 (open triangles), or PA28γ N151S (closed diamonds) were assayed for their ability to cleave LRR to detect PA28γ activity. (E) The interaction between the PA28γ and PA28γ mutant defective for proteasome activation (N151Y, N151S, G150S), (Δ245-254, P245A) and p30HA were analyzed after transient expression in 293T cells. (F) 293T cells were transfected with RL-TK-tax/rex in the presence or absence of p30 and treated with proteasome inhibitor Bortzomib at 1 micromolar for 24 hours and assayed for renilla activity.