Ab24182

Formalin-fixed paraffin sections were deparaffinized in xylene and rehydrated through an ethanol series. The sections were then immersed in 0.3% hydrogen peroxidase for 15 min to deplete endogenous peroxidase and exposed to high pressure to activate their antigenicity. Sections were then incubated with blocking solution (PBS, 3% of BSA, 0.1% Triton-X 100) at room temperature for 30 minutes. The primary antibody of TLR4 (1:20, ab22048; Abcam), MD2 (ab24182; Abcam), Myd88 (ab2068; Abcam), NF-κB (ab7970; Abcam), IL-6 (ab6672; Abcam), TGF-β (ab66043; Abcam), VEGF (ab1316; Abcam), EGF (ab115562; Abcam), and MMP2 (ab86607; Abcam) were added and incubated at 4 °C overnight. Sections were washed with PBS, incubated with FITC–conjugated goat anti-mouse secondary antibody at room temperature for 1 hour (1:400), and washed three times with PBS at room temperature for 10 minutes. Images were obtained with a fluorescence microscope (OLYMPUS DP70; Olympus Co., Tokyo, Japan).

Shanghai R&S Biotechnology Co., Ltd. (Shanghai, China) provided the vascular smooth muscle cells (VSMCs). The VSMCs underwent the culture in DMEM covering 100 U/mL of streptomycin, 100 U/mL of penicillin, and 10% fetal bovine serum (FBS) at 37° C in a 5% CO2 incubating element under humidification. Sigma (St. Louis, MO, USA) provided AngII and anti-bromodeoxyuridine (BrdU). Cell Signaling Technology (Danvers, Massachusetts) offered antibodies for P38 (8690S), P-P38 (4511), ERK (9102S), P-ERK (4695S), JNK (9252S), P-JNK (4668S). Abcam (Cambridge, MA) offered MD2 (ab24182), CD31 (ab119341), α-SMA (ab32575), Collagen III (ab-23445), PCNA (ab29), Vimentin (ab8978), 3-NT (ab191308), NOX4 (ab133303), TNF-α (ab-1392), GAPDH (ab-8245), TRITC-conjugated secondary antibody and PE-conjugated secondary antibody. Santa Cruz (CA, USA) offered SIRT1 (sc-74465). The small molecule MD2 inhibitor, L6H21, was synthesized by our lab with a purity of 98.9% [19 (link)]. L6H21 was dissolved in dimethylsulphoxide for in vitro studies.

Aortic tissue was collected, cleaned of surrounding perivascular adipose tissue, homogenized in extraction buffer (T-PER + protease inhibitor cocktail), and stored at − 80 ${}^{\circ }$ C. Measurement of overall protein concentration was performed using a BCA protein assay kit. In total, 20 μg of protein was loaded into SDS-PAGE gel with a 10% concentration followed by transfer to a nitrocellulose membrane. A solution of 5% nonfat-dry milk diluted in Tris-buffered with 1% Tween was used to block nonspecific binding sites during one hour at room temperature. Then, we probed the membranes overnight at a temperature 4  ${}^{\circ }$ C with primary mouse monoclonal antibody for TLR4 (Santa Cruz Biotechnology, sc293072; 1:1,000) and primary rabbit polyclonal antibody for MD2 (Abcam, ab24182; 1:1,000). Immunostaining was detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:10,000) for 1 hour at room temperature under constant agitation. Immunoblots were revealed by the SuperSignal West Femto Substrate (Thermo Fisher Scientific; 34095). Tissue protein levels were normalized to β-actin (Abcam; ab8227; 1:5,000) expression and quantitated by pixel densitometry using ImageJ (NIH, Bethesda, MD, USA).

Samples were dissolved in a lysis buffer (RIPA: phosphatase protease inhibitor: protease inhibitor = 100:1:1). The protein was separated with SDS‐PAGE gel and then transferred to PVDF membranes. The membranes were then incubated overnight at 4°C with the primary antibodies: anti‐p‐STING (ThermoFisher, PA5‐105674, 1:500, rabbit), anti‐STING (GeneTex, GTX33549, 1:1000, rabbit), anti‐p‐RIPK3 (Abcam, ab24182, 1:500, rabbit), anti‐RIPK3 (Abcam, ab62344, rabbit), anti‐p‐MLKL (Abcam, ab196436, 1:500, rabbit), anti‐MLKL (Abcam, Cambridge, ab243142, 1:500, rabbit), anti‐p‐PERK (Cell Signaling, mAb#3179, 1:500, rabbit), anti‐PERK (GeneTex, GTX129275, 1:1000, rabbit), GRP78 (Abcam, ab21685, 1:1000, rabbit), and anti‐GADPH (GeneTex, GTX239 1:1000, mouse). Subsequently, they were incubated for 2 h at room temperature with the HRP‐conjugated secondary antibodies. The target proteins of membranes were detected via enhanced chemiluminescence in ChemiDoc TM imaging equipment (Bio‐Rad, CA, USA).

Macrophages were treated with LPS (100 ng/ml) and 3 µg/ml gCTRP9, MD2‐neutralizing antibody (anti‐MD2; 100 ng/ml) or vehicle for 15 min. The appropriate volume of total protein extraction reagent was added to the cells. The immunoprecipitated cell extracts were incubated with an anti‐MD2 (ab24182; or anti‐TLR4 antibody 1:200; Abcam) for 1 hr, and immunoprecipitation was performed with SureBeads™ StarterKit Protein A (#1614813; Bio‐Rad, Hercules, CA) at 4℃ overnight. Treated magnetic beads were resolved by 8–12% SDS‐PAGE and transferred to PVDF membranes. Samples were immunoblotted to detect TLR4 (or MyD88) as a coprecipitated protein, and membranes were then probed with specific primary and secondary antibodies. The immune complexes were exposed in a dark box instrument after adding enhanced ECL reagent, and the AlphaEaseFC software (Kreuzbergstr, Berlin, Germany) processing system analyzed the optical density of the target band.