Inhibiting proteasome activity. Results are shown for a typical simulation, for a 20 hour period (a) Model output (k69 = 0, all other parameters as in Tables 2 and 3). (i) Level of native protein (NatP), misfolded protein (MisP), and misfolded protein bound to E3 (E3_MisP). (ii) Ubiquitin pools. (iii) Ubiquitin bound to E1 and E2 (E1_Ub and E2_Ub respectively). (iv) Cumulative number of degradation reactions for different length of ubiquitin chains bound to substrate. (v) Accumulation of aggregated protein (AggP), sequestered aggregated protein (SeqAggP) and aggregated protein bound to proteasomes (AggP_Proteasome). (vi) Available pool of proteasomes, number of proteasomes bound by substrates and number of proteasomes bound by aggregated proteins. (b) Survey of ubiquitin pools in various cell lines. Western blot analysis of cell extracts from U87MG, NIH-3T3, IMR90 and ts20 cells with an antibody raised against ubiquitin. The pool of conjugated ubiquitin (vertical line) was found to differ between cell lines with the highest levels detected in NIH-3T3 cells. Comparable levels of conjugated ubiquitin were detected in IMR90, U87MG and ts20 cells cultured at 34°C. ts20 cells cultured at 42°C for 3 hours had the least detectable levels of poly-ubiquitinated conjugates. Whereas a shift from monomeric (arrowhead) to conjugated ubiquitin (vertical line) was observed in IMR90 cells treated with proteasome inhibitor for 3 hours, no difference in the pool of monomeric ubiquitin was observed in ts20 cells treated with the inhibitor. The membrane was re-probed with an antibody directed against GAPDH which served as a loading control (boxed panel).