Hmgb1

Cells were lysed 4 h after incubation with serum-derived EVs from control and TBI patients with a lysis buffer containing protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA) and resolved in 4–20% Tris-TGX Criterion gels (Bio-Rad, Hercules, CA, USA). Protein detection was performed using antibodies to caspase-1 (Novus, Centennial, CO, USA), ASC (Santa Cruz, Santa Cruz, CA, USA), AIM2 (Santa Cruz, Santa Cruz, CA, USA), GSDMD (Novus, Centennial, CO, USA), and HMGB1 (Millipore, Burlington, MA, USA). Chemiluminescent quantification was performed using Bio-Rad Image Lab (Bio-Rad, Hercules, CA, USA) and all data were normalized to β-actin (Sigma, St. Louis, MO, USA).

Mouse and human brain extracts were prepared and Western blot analyses performed as described earlier (Sathe et al., 2012 (link)). Primary antibodies were: HMGB1 (1:1000, Millipore UK); RAGE (1:1000, Millipore); S100B (1:1000, Sigma); cyclooxygenase-2 (COX-2) (1:250, BD Bioscience, UK); tumour necrosis factor-alpha (TNF-α) (1:1000, Abcam, UK); β-actin (1:25,000, Sigma). Blots were incubated at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies (anti-rabbit or anti-mouse 1:10,000, Amersham) and ECL solution (Luminol sodium salt in 0.1 mM Tris HCl and Para-hydroxycoumarin in DMSO) were used for chemiluminescence detection. Bands were quantified using the FluorChem 8800 digital image system (Alpha Innotech, UK).

Cells were homogenized in 0.5%Triton X-100(Beyotime Biotechnology, China) with 1x Halt^™ Protease and Phosphatase Inhibitor Single-Use Cocktail, EDTA-Free (Thermo Fisher Scientific). Cell lysate was centrifuged at 12000 rpm for 20 min at 4 °C to extract clear lysates. For immunoprecipitation, incubate antibody (5 μg IgG or HMGB1)-magnetic beads protein A/G (Millipore, Germany) complex at 4 °C for 4 h with shaking. Add 50ug clear lysates to the precipitates after centrifugation. Incubate the cell lysates-antibody-magnetic beads complex with shaking at 4 °C overnight, then the beads were washed three times with lysis buffer and the precipitates were eluted with 100 μl 1 × SDS loading buffer at 100 °C for 10 min. Elutes and whole cell lysate (input) were resolved on SDS-PAGE followed by immunoblotting with antibodies.

Soluble proteins (25 µg) prepared as described in immunoprecipitation were separated by 10% SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes by reverse electrophoresis. After blocking, the membrane was stained with antibodies against TBP (1TBP18) (1∶3000 dilution, GeneTex), HMGB1 (1∶2000 dilution, Sigma), HSPA5 (1∶500 dilution, Santa Cruz), LC3 (1∶1000 dilution, Novus), H3F3B (1∶3000 dilution, GeneTex), ACTB (1∶5000 dilution, Novus), TUBB (1∶5000 dilution, Sigma) or GAPDH (1∶1000 dilution, MDBio). The immune complexes were detected using horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit (Jackson ImmunoResearch) IgG antibody (1∶10000 dilution) and a chemiluminescent substrate (Millipore).

HCT-116/HT-29 cells (2 × 10⁶ cells) were seeded in a 100-mm dish. After treatment with luteolin and/or selumetinib for 48 h, cells were collected in PBS and lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology, Beijing, China) on ice for 30 min, and lysates were harvested by centrifugal separation. Protein concentration was determined using the BCA Protein Assay Kit (Takara). Identical amounts of protein were separated using 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were clogged with 5% nonfat dry milk for 1 h at room temperature. The primary antibodies BCL-2, BAX, cleaved caspase-3, cyclin B1, p-CHK1, CDC2, XRCC1, LIG1, HMGB1, ERCC3, p-MEK1, MEK1, p-ERK1/2, ERK1/2, and GAPDH (1:1000 dilution, Sigma-Aldrich) were added after overnight incubation at 4°C. After three washes with TBST, the membranes were incubated with anti-mouse horseradish peroxidase (HRP)-linked secondary antibodies (Beyotime Institute of Biotechnology, Beijing, China) for 1 h. The intensity of protein expression was measured using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA) [31 (link),32 ].

Lysates were isolated from the whole tissue homogenates or gastric cancer cells using a Total Protein Extraction kit (Millipore, Billerica, MA, USA) and were cytoplasmic and nuclear protein extracted using a Cytoplasmic and Nuclear Protein Extraction kit (Promega, Madison, WI, USA) and were subjected to western blot analysis. Antibodies against HMGB1 (1:8,000; Sigma), LC3B, ERK1/2, phospho-ERK1/2, p38, p38, AKT, phospho-AKT, JNK, phospho-JNK (1:1,000; Cell Signaling Technology, Danvers, MA, USA), RAGE (1:200; R&D Systems), β-tubulin, albumin and lamin B (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used to develop immunoreactive signals. Densitometry was performed using AlphaImager 2200 system and Quantity software.