Nishitoh, Kadowaki, Nagai, Maruyama, Yokota, Fukutomi, Noguchi, Matsuzawa, Takeda , Ichijo
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Supplemental Materials and Methods
Cell Cultures
NSC34 cells and HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), and 100 U/ml penicillin G. Primary embryonic murine spinal cords were cultured as described (Urushitani et al. 2002). Cultured cells were infected with lentiviruses 7 days after seeding.
Plasmids and Transfection
Flag-hSOD1wt, Flag-hSOD1A4V, Flag-hSOD1G85R, Flag-hSOD1G93A, Myc-hSOD1wt, Myc-hSOD1A4V, Myc-hSOD1G85R, Myc-hSOD1G93A, hVCP, hUfd1, hNpl4, Myc-hVIMP, hDerlin-1(WT)-HA, hDerlin-1(∆C)-HA, VenusU [a short degron-, CL1 (Bence et al. 2001), tagged unstable Venus (a variant of EYFP) variant], Venus-hDerlin-1(CT1)-HA, Venus-hDerlin-1(CT2)-HA, Venus-hDerlin-1(CT3)-HA, Venus-hDerlin-1(CT4)-HA, Venus-hDerlin-1(CT5)-HA, Venus-hDerlin-1(CT6)-HA, Venus-hDerlin-2(CT)-HA, Venus-hDerlin-3(CT)-HA, and NHK (mutant form of a1-antitrypsin) were constructed in pcDNA3.0 (Invitrogen) by polymerase chain reaction. Transfection was performed with FuGENE6 (Roche) according to the manufacturer’s instructions.
Production and infection of Recombinant Adenoviruses and Lentiviruses
Recombinant adenoviruses encoding hSOD1wt, hSOD1A4V, hSOD1G85R, hSOD1G93A, Flag-hSOD1wt, Flag-hSOD1A4V, Flag-hSOD1G85R, Flag-hSOD1G93A, and mIRE1ß-Flag were generated by ViraPower™ Adenoviral (Invitrogen) according to the manufacturer’s instructions. Recombinant adenoviruses encoding HA-hASK1 was generated as described (Saitoh et al. 1998). HIV-1-based lentiviral vector system was obtained from D. Trono. Myc-mTRAF2, Venus-hDerlin-1(CT4)-HA, and Venus-HA were constructed in pRRL. Lentiviruses were produced by transient transfection into HEK293T cells as described (Zufferey et al. 1997). Viruses were concentrated 20 fold by centrifugal filtration in VirakitTM Lenti-3 (Virapur LLC). Estimated virus titers were in the range of 1 × 107 to 108 transducing units/ml. NSC34 cells were infected with recombinant adenoviruses at a multiplicity of infection (m.o.i.) of 50 or 100. Primary embryonic spinal cords were cultured in the neurogrowth medium consisting of 2% horse serum, 10 µg/mL bovine serum albumin, 10 µg/mL insulin, 26 ng/mL sodium selenite, 100 µg/mL conalbumin, 13 ng/mL progesterone, 32 µg/mL putrescine, 20 ng/mL corticosterone, 20 ng/mL triiodotyronine, 0.1 ng/mL brain-derived neurotrophic neurotrophic factor (BDNF), 10 ng/mL ciliary neurotrophic factor (CNTF), and 0.1 ng/mL neurotrophin-3 (NT3) in DMEM-F12 medium for 7 days and infected with recombinant lentiviruses for 3 days.
Antibodies
Antibodies to a1AT (MBL), SOD1 (Stressgen), GFAP (DAKO), unphosphorylated neurofilament-H (SMI32; COVANCE), p38 (Santa Cruz Biotechnology), Derlin-2 (MBL), and GFP (MBL) were purchased. Antibodies to IRE1a, PERK, and CHOP were obtained from D. Ron and F. Urano (Urano et al. 2000). Antibody to ASK1 has been described (Nishitoh et al. 2002). Peptide-specific antibodies against Derlin-1 and TRAF2 were raised in rabbits. For generation of an antibody to TRAF2, the synthetic peptide SRQHRLDQDKIEAC was used as an immunogen and synthetic peptide. For generation of an antibody to Derlin-1, synthetic peptides QFLYRWLPSRRGG and GRHNWGQGFRLGDQ were used as an immunogen. The specificity of the purified polyclonal antibodies (immunoguloblin G) was confirmed by IB or IP (Supplementary Fig. 3).
Immunoblotting analysis
Immunoblotting analysis has been described (Tobiume et al. 2001). Blots were probed with antibodies to CHOP, SOD1, Derlin-1, Derlin-2, MAP2, p38, p97, Flag (Sigma), HA (Roche), and Myc.
Immunocytochemistry
HEK293 cells were plated onto 13 mm-diameter cover grass, transiently transfected, and cultured overnight. Primary spinal cords were plated onto 13 mm-diameter cover grasses, cultured for 7 days, and infected with lentiviruses for 72 h. After a brief wash at room temperature (RT) in PBS, cells were fixed in 4% paraformaldehyde (PFA) in PBS for 30 min at RT. After several washes in PBS, cells were permeabilized and blocked in PBS containing 0.2% Triton X-100 and 3% bovine serum albumin (BSA) for 1 h at RT. They were then incubated with antibodies to HA, unphosphorylated neurofilament-H (SMI32), and GFAP in PBS containing 0.2% Triton X-100, and 3% BSA for 16 h at 4 oC. After several washes in PBS, samples were subsequently with corresponding fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories), mounted on the slide grass, and analyzed by a microscope.
References
Bence, N.F., Sampat, R.M., and Kopito, R.R. 2001. Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292(5521): 1552-1555.
Nishitoh, H., Matsuzawa, A., Tobiume, K., Saegusa, K., Takeda, K., Inoue, K., Hori, S., Kakizuka, A., and Ichijo, H. 2002. ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16(11): 1345-1355.
Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. 1998. Mammalian thioredoxin is a direct inhibitor of apoptosis signal- regulating kinase (ASK) 1. EMBO J 17(9): 2596-2606.
Tobiume, K., Matsuzawa, A., Takahashi, T., Nishitoh, H., Morita, K., Takeda, K., Minowa, O., Miyazono, K., Noda, T., and Ichijo, H. 2001. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep 2(3): 222-228.
Urano, F., Wang, X., Bertolotti, A., Zhang, Y., Chung, P., Harding, H.P., and Ron, D. 2000. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 287(5453): 664-666.
Urushitani, M., Kurisu, J., Tsukita, K., and Takahashi, R. 2002. Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis. J Neurochem 83(5): 1030-1042.
Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L., and Trono, D. 1997. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15(9): 871-875.