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. 2002 Jun 1;16(11):1345–1355. doi: 10.1101/gad.992302

Figure 4.

Figure 4

Requirement of ASK1 for ER stress-, proteasome inhibitor-, and polyQ-induced JNK activation and cell death in neurons. (A,B) Lack of ER stress-induced cell death in ASK1−/− primary neurons. ASK1+/+ neurons (○) and ASK1−/− neurons (●) were treated with the indicated concentration of thapsigargin or tunicamycin for 16 h. The graphs show the cell viability determined by MTT assay as described in Materials and Methods. (C) Reduction of proteasome inhibitor-induced cell death in ASK1−/− primary neurons. ASK1+/+ neurons (open bar) and ASK1−/− neurons (solid bar) were treated with 0.1 μM MG132 for 16 h. The graph shows the cell viability determined by MTT assay. Data are means (± SE) of three independent experiments derived from independent embryos (A–C). (*) P < 0.05 relative to control; significance calculated by Student's t-test. (D) Lack of polyQ-, proteasome inhibitor-, and ER stress-induced JNK activation in ASK1−/− primary neurons. ASK1+/+ neurons and ASK1−/− neurons were infected with the indicated m.o.i. of Ad-β-gal or Ad-Q79 for 48 h, or treated with 2 μM thapsigargin for 30 min, or 10 μM MG132 for 2 h. JNK3 was immunoprecipitated by anti-JNK3 monoclonal antibody and subjected to immunecomplex kinase assay as described in Materials and Methods. (Top) IVK for JNK3 activity. Expressions of JNK3 (middle) and Flag-Q79 (bottom) in the same lysate are shown. Kinase activity relative to the amount of JNK3 protein was calculated, and activity is shown as fold increase relative to that of JNK3 from unstimulated cells. (E) Time-course analysis of polyQ-induced cell death in ASK1+/+ and ASK1−/− primary neurons. ASK1+/+ neurons (○, ▵) and ASK1−/− neurons (●) were infected with Ad-Q79 or Ad-Q35 at m.o.i. 100 for the indicated time periods. The graph shows the cell viability determined by MTT assay as described in Materials and Methods. Data are means (±SE) of 10 independent experiments derived from independent embryos.