Biochemi system

miRNAs and their corresponding target genes were inserted into vector GATEPEG100. All constructs were transformed into Agrobacterium tumefaciens strain GV3101. N. benthamiana plants, seeded directly in pots, were maintained in an incubator at 24°C with 12 h light/12 h dark cycle. A. tumefaciens cultures were grown in liquid LB medium with selection [82] (link). After 40 h, leaves were harvested, and protein extraction was performed [83] (link). Proteins were separated on 10% SDS–PAGE gels and transferred onto nitrocellulose membranes (Millipore, Billerica, MA). Membranes were blocked using 5% milk in 1×TBST and then incubated with Anti-FLAG (DYKDDDDK) Antibody (#635691, Clontech, Mountain View, CA) followed by a secondary horseradish peroxidase (HRP)-conjugated goat anti-Rabbit polyclonal antibody (#A0504, Sigma, St. Louis, MO) according to the manufacturer's recommendations. Reactive species were visualized using SuperSignal West Pico Chemiluminescent Substrate (#34087, Pierce, Rockford, IL) and imaging using a Biochemi system (UVP, Upland, CA).

C6 cells were plated in a 6-well cell culture plate (TPP 35 mm tissue culture plate, MidSci, St. Louis, MO, USA) and allowed to adhere overnight. The following day, cells were treated with the pan-caspase inhibitor zVAD-fmk (FMK001, R&D systems, Minneapolis, MN, USA) at a concentration 100 µM or with 100 µM of resveratrol. Two hours after pretreatment, cells were UV-irradiated (UV-C, 254 nm) for 5 min using a 3UV transilluminator (BioChemi System, UVP, Upland, CA, USA) as described by us previously [18 (link)]. 12 h after UV-irradiation, cells were harvested for immunoblot analysis, caspase activity measurements or stained for NFT formation using Thioflavin S.