Ab18256

Tissues were homogenized in radioimmunoprecipitation assay buffer (20 mM tris,150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM tetrasodium pyrophosphate, 1 mM β-glycerophosphate, and 1 mM sodium orthovanadate) containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA) using a Precellys sample lyzer (Bertin Technologies, Montigny le Bretonneux, France). Western blots were performed using standard procedures using antibodies against HMGB1 (1:1000, ab18256, Abcam, Cambridge, UK), phospho-AKT S473 (1:1000, CST 4060, Cell Signaling Technology, Danvers, MA, USA), total AKT (1:1000, CST 9272, Cell Signaling Technology, Danvers, MA, USA), hemagglutinin (HA) (1:1000, CST 3724 Cell Signaling Technology, Danvers, MA, USA), PPARγ (1:1000, C26H12, Cell Signaling Technology, Danvers, MA, USA), Myc-tag (1:1000, CST 2276, Cell Signaling Technology, Danvers, MA, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1: 2000, ab181602, Abcam, Cambridge, UK), used as a loading control.
Detection of high–molecular weight proteins was performed using capillary electrophoresis (Wes, SimpleProtein, San Jose, CA, USA) using antibodies against FAS (CST 3189, Cell Signaling Technology, Danvers, MA, USA), ACC (CST 3662, Cell Signaling Technology, Danvers, MA, USA), and ACLY (ab40793, Abcam, Cambridge, UK).

Corneas (n = 4) were harvested at 2, 4, 7, 14, 28 days and ground and then homogenized in RIPA lysis buffer containing a mixture of protease and phosphatase inhibitor. Lysates were then centrifuged at 12,000 × g for 10 min at 4°C and supernatants were harvested, and the concentration was valued with a bicinchoninic acid assay kit. Subsequently, samples were mixed with 5X SDS-PAGE loading buffer and boiled for 10 min at 100°C. Equivalent amount of protein were run in a 10–12% SDS-PAGE gel and then transferred to PVDF membrane. Membranes were cut properly and immersed in 5% milk for 2 h at room temperature, and then incubated overnight at 4°C with primary antibodies for HMGB1 (1:500 dilution, ab18256, Abcam), VEGF (1:1000 dilution, ABS82, Sigma Aldrich), TNF-α(1:1000 dilution, ab6671, Abcam), Fibronectin (1:1000 dilution, sc-9068, Santa Cruz Biotechnology), GAPDH (1:1000 dilution, ab181602, Abcam) and β-actin (1:1000 dilution, no4967, Cell Signalling Technology). On the following day, secondary antibody (1:5000 dilution) incubation was managed for 2 h at room temperature. Protein expression was measured by the Odyssey® Sa Two-colour infrared laser imaging system (Licor, United States). The images were analyzed by Image-Pro Plus 6.0.

ChIP assay was conducted using the EpiQuick Tissue Chromatin Immunoprecipitation Kit (Epigentek, Brooklyn, NY) according to the manufacture's instruction and rabbit polyclonal antibody against HMGB1 (ab18256, Abcam). The immunoprecipitated chromatin was analyzed in duplicate by PCR using the primers (Mo-Gsto1-ChIP-2-F: 5′-AAAGTGGACGAAACCCTTGA-3′ and Mo-Gsto1-ChIP-2-R: 5′-CACACACGCATGTGACAGAA-3′) for mouse Gsto1 promoter. The binding site was identified using Transcription Factor Search web site (http://www.cbrc.jp/research/db/TFSEARCH.html). After cloned the PCR products into pCR2.1 vector using TA cloning kit (invitrogen), the sequence of PCR products was confirmed.

The cells were seeded on 24-well plates (Thermo Scientific, USA) at a density of 1 × 10⁴ cells per well. After 24 h, the medium with serum was removed and the cells were incubated with the nanoparticles (0.5 μg/mL BODIPY) diluted in cell media for 6 h. The cells were washed with phosphate-buffered saline and the cells further incubated with anti- nuclear HMGB1protein antibody (Abcam, ab18256, 1:50) for 2 h at 4 °C. Subsequently, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (Abcam, ab150077, 1:500) for 1 h. The cells were exposed to irradiation (650 nm, 0.1 W cm⁻², 2 min). The secretion of nuclear HMGB1protein inside the cells was assessed using CLSM (LSM-800, ZEISS, Germany). DAPI: λ_ex = 410 nm, λ_em = 506 nm, nuclear HMGB1protein specific antibody: λ_ex = 488 nm, λ_em = 525 nm.