Figure 3.

Modulation of the human 26S proteasome by Ecm29 overexpression. A, effect of Ecm29 overexpression on 26S proteasome integrity. Shown are quantitative immunoblot analyses of purified proteasomes (left) and cell lysates (right) in the presence or absence of overexpressed Ecm29. 293^Rpn11-HTBH cells were transiently transfected with FLAG–Ecm29 either treated with H₂O₂ or untreated as a control. Quantitative Western blotting was performed with antibodies against Ecm29, Rpt6 (19S), and α7/Pre10 (20S). B, effect of Ecm29 overexpression on 26S proteasome activities. The proteasomal proteolytic activities were determined by in-solution peptidase activity assays with three fluorogenic peptide substrates: SUC-LLVY-AMC (LLVY); SUC-LLE-AMC (LLE), and SUC-ARR-AMC (ARR). The activities were normalized to Rpn11 (a 19S subunit) in each sample. Data were from three experiments. The numbers under the immunoblot bands represent quantitative measurements using a Fuji LAS4000 scanning system.