Enhanced chemiluminescence reagent

Total cell extracts were prepared using the TCA method (Keogh et al., 2006 (link)). Proteins were separated on 8–15% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were probed with specific antibodies, and immuno-reactivity was detected using enhanced chemiluminescence reagent (Elpis Biotech, Korea). The primary antibodies were anti-Sir2 (1:200, Santa Cruz, Dallas, TX), anti-FLAG (1:1000, Sigma, Saint Louis, MO), anti-Myc (1:1000, Santa Cruz), anti-GAPDH (1:10,000, Acris, Germany), anti-AcH4K16 (1:2000, Upstate, Lake Placid, NY), and anti-H4 (1:0000, Millipore). Band density trace and quantification were determined using ImageJ (National Institutes of Health).

HEK 293T cells (ATCC, CRL-3216) were transfected by using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s recommendations, harvested 2 days after transfection, and maintained at −80°C. Cell lysates and culture supernatants were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA). The membrane was incubated with a 1:1,000-diluted sample of rabbit polyclonal IgG (Sino Biological, BDA, Beijing, China, catalog no. 40069-RP02) to detect SΔER, SΔTM, S1, S2, and RBD overnight at 4°C in blocking buffer (PBS [Thermo Fisher Scientific], 5% skim milk [BD Bioscience, San Jose, CA], 0.05% Tween 20 [Sigma, St. Louis, MO]), followed by washing. The blot was further incubated in a blocking buffer with 1:5,000 dilutions of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Southern Biotech, Birmingham, AL) for 1 h at 22°C and then washed. Detection was performed with the enhanced chemiluminescence reagent (ELPIS-Biotech, Daejeon, South Korea).