Wide mini sub cell gt system

After 24 h of treatment, total protein was isolated from cells by RIPA lysate with protease inhibitor and phosphatase inhibitor. After determining the protein concentration by the BCA protein assay kit, 20 μg protein was added into each well of the vertical electrophoresis tank and separated by 10% SDS-PAGE. Subsequently, the protein was transferred onto the PVDF membrane. The Power Pac basic power supply, Wide mini-sub cell GT system, Trans-Blot SD Semi-Dry electrophoretic transfer tank were all provided by Bio-Rad (Bio-Rad Laboratories, Inc., Hercules, CA, USA). After being blocked by 5% skim-milk for 2 h, then the incubation of primary antibodies (TNF-α, p65, p-p65, IκBα, p-IκBα, GBP2, IRF1, SAMHD1, p-SAMHD1, β-actin, GAPDH, 1:2000) was performed overnight at 4 °C. After washing by TBST three times for 30 min, the secondary antibodies (anti-rabbit IgG HRP-linked antibody, 1:2000) were incubated for 1 h at room temperature. After TBST wash, the ECL chromogenic substrate was used to detect specific bands in Image Quant LAS 500 (GE HealthCare Technologies Inc., Chicago, IL, USA). The gray value was estimated by Image J software (National Institutes of Health, Bethesda, MD, USA) normalized to β-actin.

Native agarose gels were prepared at 1% (w/v) and submerged in TAE running buffer, comprising of 40 mM Tris-acetate and 1 mM EDTA (pH 8). The gel loading buffer consisted of 30% (v/v) glycerol and 0.25% (w/v) bromophenol blue in DNase-free water. The loading buffer was combined with ~ 500 ng of drug-treated native (un-cleaved) pUC19 DNA sample in a 1:5 ratio, made up to a final volume of 6.25 μL. The 1% (w/v) agarose gel were cast and run on a Bio-Rad Wide Mini-Sub Cell GT System with 6.25 μL drug-treated plasmid DNA samples or untreated plasmid DNA sample (DMF solvent control) and 5 μL of an Axygen Biosciences ready-to-use 1 kb DNA molecular weight ladder. Gel electrophoresis was carried out at 5.5 V/cm for 3 h, followed by staining in 1 x GelRed stain (diluted in 1 x TAE buffer, pH 8) for at least 30 min. The gel was visualised under UV light with a BioRad Gel Doc 2000 imager and compound-induced apparent molecular weight changes of the plasmid DNA were analysed against the 1 kb ladder standard, using BioRad Quantity One gel imaging software.

Agarose gel electrophoresis
was used to analyze the folding of the DNA origami unit as well as
the formation of DNA origami dimers. A 2% (w/v) agarose gel was prepared
in 1× TAE buffer containing 11 mM MgCl₂ for the gel
at pH 8.2, whereas the 2% (w/v) agarose gel was prepared in 45 mM
MES and 25 mM Tris containing 11 mM MgCl₂ for the gel at
pH 6.0. Both gels were stained with ethidium bromide (final concentration
of 0.46 μg mL^–1). Depending on the sample
and the type of gel, the sample volume was 10–18 μL and
the DNA origami concentration was 11.1/15.0 nM (DNA origami units)
or 5.4/5.7 nM (DNA origami dimers). A gel loading dye solution was
added to the samples at a ratio of 1:5 before loading the samples
in the gel pockets. The gel was run for 45 min at a constant voltage
of 95 V using a BioRad Wide Mini-Sub Cell GT System and a BioRad PowerPac
Basic power supply while keeping the gel electrophoresis chamber on
an ice bath. For the gel at pH 8.2, the running buffer was 1×
TAE buffer supplemented with 11 mM MgCl₂, whereas 45 mM
MES and 25 mM Tris supplemented with 11 mM MgCl₂ was used
as running buffer for the gel at pH 6.0. After the run, the gel was
visualized by ultraviolet light using a BioRad Gel Doc XR+ documentation
system.