Streptavidin hrp antibody

EMSA was performed using the Lightshift Chemiluminescent EMSA Kit (Thermo Fisher)^{37 (link)}. D-loop mtDNA with oxidized guanine was synthesized by IDT. The 100 μM stock of Ox-DNA was diluted twice at 1:100 and once at 1:10. NLRP3 protein extract was serially diluted twofold from 0.225 μg/μL to be used in ten reactions. Each 20 μL reaction consisted of NLRP3 protein extract or purified protein, 1 × 10⁻⁵ μM oxidized mtDNA, and binding buffer (50 mM Tris, 100 mM NaCl, 2 mM MgCl₂, 2 × 10⁻⁵ mg/ml sonicated salmon sperm DNA, 12% glycerol, and pH 7.4). Samples were incubated at 4 °C overnight and run with native conditions on 4–20% Tris/glycine gels. Protein-DNA complexes were transferred to the Biodyne B membrane and UV-crosslinked. Membranes were blocked in 5% BSA for 1 h, washed three times for 10 min, and probed for anti-biotin with streptavidin-HRP antibody (BD Pharmingen). After three additional washes, membranes were exposed to an anti-mouse secondary antibody for 1 h, washed three times for 10 min, and subjected to chemiluminescence. The intensity of the HRP reaction was captured on an x-ray film (KODAK) and developed. Subsequently, membranes were stripped with 25 mM glycine, 1% SDS pH 2.4, and reprobed for human NLRP3 pyrin domain (Adipogen)^{2 (link)}.

On 26th day of the treatment tumor tissues were collected from mice. Tumor tissues were fixed in 10% formalin, processed for paraffin embedding, and cut into 5 μm sections. Each section was stained with H & E stain visualized under a light microscope and photographed. Immunohistochemistry of tumor tissue, for the assessment of VEGF or CD31, was performed by incubating the sections with mouse anti-human VEGF antibody (BD Biosciences) or rabbit anti-mouse CD31 antibody (Abcam, Cambridge, UK) at 4°C overnight. Sections were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Southern Biotech, Birmingham, AL) or biotinylated polyclonal anti-rabbit antibody (Vector Laboratories, Burligame, CA), followed by streptavidin-HRP antibody (BD Biosciences). Sections were then developed with diaminobenzidene (DAB; Dako, Carpinteria, CA) and counterstained with Meyer's hematoxylin.

For each protein, 18 μL at >1 mg/mL was incubated with 2 μL of 1 μM biotinylated DNA for 1 h at 4 °C. During the reaction, an Invitrogen™ Novex™ WedgeWell™ 4–12%, Tris-Glycine gel was pre-run for 30 min at 225 V. Samples were loaded, and the gel was run for 30 min at 225 V. Samples were transferred to a PVDF membrane, blocked using 2.5% BSA in TBST, and probed with a Streptavidin-HRP antibody (BD BioScience) at a 1:2000 dilution in 2.5% BSA in TBST. The membrane was analyzed using the iBright 1500 Imaging system, then stripped with 25 mM glycine, 1% SDS pH 2.4, and rocking at room temperature for 40 min. The blot was rinsed with deionized H₂O, and the stripping buffer was removed by washing with TBST for 45 min, changing the buffer every 5 min. The membrane was then blocked with 2.5% BSA and probed with a pyrin-targeting primary antibody (AdipoGen) diluted 1:10,000 in 2.5% BSA in TBST. The membrane was washed with TBST for 5 min three times, incubated with an anti-mouse HRP secondary, and analyzed again on the iBright 1500 Imaging system. Subsequently, the membrane was blocked and reprobed with an anti-NACHT rabbit monoclonal antibody (Cell Signaling) diluted 1:2000 in 2.5% BSA in TBST. The membrane was washed with 1x TBST for 5 min three times, incubated with an anti-rabbit secondary, and then analyzed on the iBright 1500 Imaging system.