Anti hmgb1 antibody

Proteins were extracted from left ventricular myocardium with RiPA Buffer (Thermo Scientific) and protease inhibitor was added. Protein concentration was determined by BCA method (Solarbio, Beijing, China). Proteins were taken at 50 μg and denatured at 100°C for 5 mins. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China) and then transferred to a polyvinylidene fluoride (PVDF) membrane by electrophoretic equipment (Bio-Rad, CA, USA). After being blocked with 3% BSA for 2 hours at room temperature, the membranes were incubated with anti-PI3K antibody, anti-phospho-PI3K antibody, anti-eNOS antibody, anti-phospho-eNOS (1 : 1000, Bioss, Beijing, China), anti-COX-2 antibody, anti-HMGB-1 antibody (1 : 1000, Proteintech, Chicago, IL, USA), and anti-ECE-1 antibody (1 : 2000, Signalway Antibody, College Park, MD, USA), primary antibodies overnight at 4°C. Next day, after being washed with TBST for 3 times, the membranes were incubated with HRP-labeled goat anti-rabbit IgG (1 : 25000, 1 h, Zhongshanjinqiao, Shanghai, China) at room temperature. After being washed with TBST for 3 times, the membranes were visualized by Pierce™ ECL Western Blotting Substrate and imaged using FluorChem E Imaging System (Protein Simple, USA), which were normalized to β-actin as an internal control.

Cell surface expression of CRT and release of HMGB1 was detected by immunofluorescence. For CLSM observation, LLC cells were seeded into 24-well plates (2 × 10⁴ cells/well), and cultured with 1.0 mL DMEM medium for 12 h. These cells were treated as follow: untreated as control group, NIR laser, UCMS@Pep, UCMS@Pep-RB + NIR laser at an equal amount of nanoparticle (100 µg/mL) for 6 h incubation. Then the NIR laser irradiation was performed for the groups of NIR laser and UCMS@Pep-RB + NIR laser, followed by another 12 h incubatioin. Thereafter, the cells were fixed with paraformaldehyde for 15 min, blocked by 2 % BSA buffer for 1 h, and incubated with anti-calreticulin antibody (Proteintech, China) or anti-HMGB1 antibody (Proteintech, China) overnight at 4 °C. Further, the cells were labeled with Alexa Fluor 488-conjugated secondary antibody for 1 h at room temperature. Next the cells were stained with phalloidin (5 µg/mL) for 1 h, and further stained with DAPI (300 nM) for 15 min. Last, the images were captured by CLSM at excitation wavelengths of 488 nm for CRT or HMGB1, 593 nm for phalloidin and 405 nm for DAPI.

Fluorescein isothiocyanate (FITC)-labeled human recombinant histone H3 (2 μg, Rockland Immunochemicals Inc., Limerick, PA, USA) was mixed with glycated HMGB1 (2 μg) or human recombinant HMGB1 in Tris-buffered saline (100 μL) and incubated at 4 °C for 12 h. The mixture solution was immunoprecipitated using an anti-HMGB1 antibody (Proteintech) and protein A/G agarose (Santa Cruz) for 3 h at 4 °C. Precipitates were collected by centrifugation and washed five times with lysis buffer. The fluorescence intensity (A₅₂₀) of histone H3 in the precipitate solution was measured using a fluorescence microplate reader (Thermo Fisher).