Figure 2.

Expanded polyQ activates the IRE1–TRAF2–ASK1–JNK pathway. (A) ER stress-induced activation of ASK1–JNK pathway. PC12 cells (1 × 10⁶) were treated with 20 μM thapsigargin (Tg) or 10 μg/mL tunicamycin (Tn) for the indicated time periods. Cells were then lysed and immunoblotted with antibodies to phospho-ASK1 (P-ASK1) and phospho-JNK (P-JNK). Membranes were reprobed with antibodies to ASK1 and JNK as loading controls. The asterisk in the phospho-ASK1 panel denotes a nonspecific band detected. (B) ER stress-dependent endogenous interaction between ASK1 and TRAF2 in PC12 cells. Cell lysates from PC12 cells treated with 20 μM thapsigargin or 10 μg/mL tunicamycin for the indicated time periods were immunoprecipitated with anti-TRAF2 (IP: TRAF2), anti-ASK1 (IP: ASK1), or nonimmune antiserum (IP: Cont.). Coimmunoprecipitated ASK1 with TRAF2 was detected by immunoblotting with anti-ASK1. Presence of ASK1 and TRAF2 was confirmed by immunoblotting using the same lysates. (C) ER stress-induced IRE1–TRAF2–ASK1 complex in transfected 293 cells. The 293 cells were transiently transfected with pcDNA3–HA–IRE1β (1.2 μg), pcDNA3–Flag–TRAF2 (0.3 μg) and pcDNA3–myc–ASK1 (0.5 μg) in the indicated combinations. After 12 h, cells were treated with 20 μM thapsigargin for 20 min, immunoprecipitated with anti-HA (12CA5), and immunoblotted (WB) with anti-myc (9E10) and anti-HA (3F10) antibodies. The appropriate expressions of Flag–TRAF2 and Myc–ASK1 were confirmed in the same lysates. (D) Activation of ASK1–JNK pathway by pathogenic polyQ. PC12-Q79 and PC12-Q14 cells were lysed at each time point after removal of Tet and analyzed by immunoblotting with antibodies to phospho-ASK1 (P-ASK1) and phospho-JNK (P-JNK). Membranes were reprobed with antibodies to ASK1 or JNK as loading controls. The asterisk in the phospho-ASK1 panel denotes a nonspecific band. Expression of Flag–Q79 was verified by immunoblotting with anti-Flag antibody. Immunoreactive bands of Q79 were detected at the top of the stacking gel accompanied by a smearing pattern (bottom). (E) PolyQ-dependent endogenous interaction between ASK1 and TRAF2 in primary neurons. Cell lysates from primary neurons (1 × 10⁷), infected with Ad-β-gal or Ad-Q79 at m.o.i. 100 for 48 h, or treated with 20 μM thapsigargin for 20 min, were immunoprecipitated with anti-TRAF2 (IP: TRAF2). Coimmunoprecipitated ASK1 with TRAF2 was detected by immunoblotting with anti-ASK1. Activation of IRE1 was confirmed by IP–WB with anti-IRE1α antiserum using the same lysates (bottom). (P-IRE1) Autophosphorylated IRE1.