Anti sting

The proteins were extracted and separated by 10% SDS-PAGE. After being transferred onto polyvinylidene fluoride membranes, the membrane was transferred for 2 h at 4 °C and blocked with 5% non-fat milk-TBST, shaking on a room temperature shaker for 2 h. After blocking and washing the polyvinylidene fluoride membrane with washing solution, then the primary antibody was added. The primary antibodies used in this study were as follow: anti-Caspase-1 (1:1000; Abcam, Cambridge, MA, USA); anti-GSDMD (1:1000; Abcam); anti-NLRP3 (1:1000; Abcam); anti-CENPM (1:2000, Abcam) anti-E-cadherin (1:2000, Abcam); anti-N-cadherin (1:2000, Abcam); anti-vimentin (1:2000, Abcam); anti-cGAS (1:1000; Abcam); anti-p-STING (1:1000; Invitrogen); anti-STING (1:1000; Abcam); anti-β-actin (1:2000; Abcam). The membrane was incubated with shaking at 4 °C overnight. Then adding horseradish peroxidase-labeled goat anti-rabbit IgG (1:5000; Abcam), the membrane was incubated at room temperature with shaking for 1 h. Washing the membrane with TBST 3 times, we performed chemiluminescent detection, exposure, and development to analyze the data. The internal reference was β-actin.

Animals were deeply anesthetized with isoflurane and perfused through the ascending aorta with PBS (phosphate-buffered saline), followed by 4% paraformaldehyde with 0.1% picric acid in 0.16 M phosphate buffer. Spinal cord (30 μm, free-floating) was cut in a cryostat. The sections were first blocked with 1% BSA + 0.1% Triton X 100 for 1 h at room temperature, and then incubated overnight at 4 °C with the primary antibodies. The following primary antibodies were used: anti-STING (1:100, Abcam), anti-NeuN (1:200, Abcam), anti-GFAP (1:200, Cell Signaling Technology), and anti-IBA-1 (1:200, Abcam). After rinsing three times with PBS, the sections were incubated with fluorescence-labeled secondary antibody for 1 h. Images were collected using a fluorescence microscope (Olympus, Japan).

Animals were sacrificed under deep isoflurane anesthesia. The dorsal horn of spinal cord segments was removed rapidly and snap-frozen in liquid nitrogen. Samples were mechanically homogenized in ice-cold radioimmune precipitation assay buffer containing phenylmethanesulfonyl fluoride (Abcam, Cambridge, UK). The protein content was determined using the bicinchoninic acid assay method. The loading and blotting of an equivalent number of total proteins were verified using a membrane with monoclonal mouse anti-β-actin antibody (1:5000; Sigma-Aldrich). The samples were resolved on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with polyclonal rabbit antibodies against anti-STING (1:1000, Abcam), TBK1 (1:1000, Cell Signalling Technology, Danvers, USA), p-TBK1 at Ser-172 (1:1000, Cell Signalling Technology), IRF3 (1:1000, Cell Signalling Technology), p-IRF3 at Ser-396 (1:1000, Cell Signalling Technology), p-ERK (1:1000, Cell Signalling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:2000, Jackson Immuno Research). The membrane-bound secondary antibodies were visualized with enhanced chemiluminescence (Thermo Scientific, Rockford, IL, USA) and quantified with Image-Pro Plus software (Media Cybernetics Inc).