Tbs tween 20 buffer

TS/A cells were electrotransfected with gWiz Blank plasmid as described above using EP1 and EP2 pulse protocols. The cells were then seeded into low-attachment 6-well plates (Corning Incorporated, Corning, NY, USA) and incubated for 9 hours. After incubation, the cells were lysed using RIPA buffer in accordance with the manufacturer’s protocol (Santa Cruz Biotechnology Inc., Dallas, TX, USA). For the preparation of crude lysate, the final centrifugation step was excluded. Total protein content was determined using a Pierce BCA Protein Assay Kit (Fisher Scientific) then adjusted with RIPA buffer. Twenty-five µg or 40 µg of total protein (for cleared and crude lysate, respectively) per well was separated in a 10% polyacrylamide gel, transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA), blocked for 1 hour in 5% milk in TBS Tween-20 buffer (Fisher Scientific) and incubated overnight at 4° C with primary antibodies: rabbit anti-DAI/ZBP1 or rabbit anti-β-actin as a loading control (Fisher Scientific). After washing with TBS Tween-20 buffer, the membrane was probed with Alexa Fluor 680 goat anti-rabbit secondary antibody (Fisher Scientific) for 45 min at room temperature, washed and protein bands were visualized with Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, NE, USA). The band intensity was quantified using Image J software [97 (link)].

Roswell Park Memorial Institute 1640 medium (RPMI-1640), fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Hyclone Laboratories (CA, USA). Cell counting kit-8 (CCK-8) and dimethyl sulfoxide (DMSO) were obtained from Dojindo Laboratories (Kumamoto, Japan) and Sigma-Aldrich (St. Louis, MO, USA), respectively. FITC annexin V Apoptosis Detection Kit and propidium iodide (PI) were purchased from BD Biosciences (San Jose, CA, USA). Radioimmunoprecipitation assay buffer, protease and phosphatase inhibitor cocktail, BCA protein assay kit, 4–12% bis-tris plus gels, nitrocellulose membranes, TBS Tween 20 Buffer, Starting Block T20 Blocking Buffer, enhanced chemiluminescence kit, and the following antibodies: caspase-9, cleaved caspase-9, PARP, cleaved PARP, Bcl-2, β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibody were acquired from Thermo Fisher (Rockford, IL, USA). Antibodies against caspase-3, cleaved caspase-3, AMPKα, Akt, and phospho-Akt were purchased from Cell Signaling (Danvers, MA, USA).