Bocd fmk

PC12 rat pheochromocytoma cells were maintained in DMEM supplemented with L-glutamine and 10% FBS at 37 °C in humidified condition of 5% CO₂/air. In some experiments, the cells were incubated with medium containing the chemical chaperone 4-PBA (TCI), the UPR activator tangeretin (TCI), pan-caspase inhibitors (zVAD-fmk from MBL and BocD-fmk from Abcam), an allosteric inhibitor of RIPK1 (necrostatin-1; Sigma), and JNK inhibitor (SP600125; LCL). 3 × FLAG-tagged human wt and Q343R mutant TDP-43 expression vectors were kindly gifted from Dr. Koji Yamanaka (Department of Neuroscience and Pathobiology, Nagoya University, Japan)52 (link). Plasmids were introduced into the cells using the FuGENE 6 (Roche) according to the manufacturer’s protocol. For RNAi-mediated gene silencing, the cells were transfected with target-specific or control scrambled siRNA using Lipofectamine RNAiMAX (Life Technologies). The following small interfering RNAs (siRNAs) were used: rat Hrs siRNA#1, 5′-CCAUCAAGAAGAAGGUCAAUGAUAA-3′; rat Hrs siRNA#2, 5′-UUAUCAUUGACCUUCUUCUUGAUGG-3′; and rat RIPK1 siRNA, 5′-GCGCUGAGUACAAUGAGGCUCUCUU-3′. Scrambled control siRNA (sc-37007) was purchased from Santa Cruz Biotechnology.

Mouse monoclonal anti-α-Tubulin, goat polyclonal anti-actin (C-11) and rabbit polyclonal anti-CD40 (N-16) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal anti-caspase-3 and anti-RIP1 antibodies were obtained from Cell Signaling Technology. Mouse monoclonal agonistic anti-CD40 (Clone # 82111) antibody was purchased from R&D Systems (Minneapolis, MN, USA). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Recombinant human sCD40 ligand (CD40L) was obtained from Peprotech (Rocky Hill, NJ, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), necrostatin-1 and 1-MT were purchased from Sigma-Aldrich. Boc-D-FMK was purchased from Abcam (Toronto, ON, Canada). GSK'872 and necrosulfonamide were purchased from Millipore (Etobicoke, ON, Canada).

Homoharringtonine (Omacetaxine®) was provided by Teva Pharmaceutical Industries Ltd. Bortezomib was purchased from Chemietek (Indianapolis). BOC-D-fmk was purchased from Abcam. All agents were formulated in DMSO and stocked in − 80 °C for in vitro use.