Scrambled control sirna sc 37007

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.

CTSB was overexpressed in Nthy-ori-3-1 and 8505C cells cultured in 3 mL media of 6-well plates at a density of 4 × 10⁵ cells per well. After 16 h, 2 μg of pCS4-3xMyc-CTSB plasmid was transfected using Lipofectamine2000 (Invitrogen, CA) according to manufacturer’s guidelines. CTSB expression was knocked down using siRNA in SNU790 and TPC-1 cells. Both siCTSB (sc-29238) and a scrambled control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Cells were seeded in 6-well plates at a density of 4 × 10⁵ cells per well. After 16 h, transfection was performed using the TransIT-X2^® Transfection kit (Mirus, MIR6000, Madison, WI, USA), according to manufacturer’s instructions. After 48 h, transfected cells of plasmid or siRNA were harvested.