Ab47093

To examine protein expressions of TLR4, NF-κB (p65), IκB-α and IL-1β in aortic tissues, aortas were removed and cleaned of adventitia from experimental mice. These tissues were washed with PBS and lysates were prepared by lysis buffer. Nuclei and cytoplasmic debris were pelleted at 14,000 × g for 20 min, and supernatants were collected. The protein content was determined by Bradford assay. Equal amounts of proteins (30 μg protein/lane) were electrophoresed using 10% sodium dodecyl sulfate polyacrylamide gels in a Tris/HCl buffer system, followed by electrophoretic transfer to a polyvinylidene difluoride microporous membrane (BioRad, Hercules, PA, USA). Subsequently, the membranes were incubated with the appropriate primary antibodies overnight at 4°C: rabbit anti-mouse β-actin (abcam, ab6276, 1:5000), rabbit anti-mouse TLR4 antibody (abcam, ab47093, 1:1000), rabbit anti-mouse IκB-α (abcam, ab32518, 1:1500) rabbit anti-mouse NF-κB p65 (cell signaling technology, 3037, 1:1000) and IL-1β (abcam, ab106035, 1:1000). Following 3 washes with TBST buffer, immunodetection was accomplished using appropriate horseradish peroxidase-linked secondary antibodies (KPL, 074–1516) and enhanced chemiluminescence system. Protein band densities were quantified by Image-J software (NIH).

Sirius red staining (connective tissue staining) kit was purchased from Abcam (ab150681, Cambridge, UK). Trizol was obtained from Life Technologies (Waltham, USA). SYBR® Premix Ex Taq™ II (RR820A) and PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A) were from TaKaRa Biotechnology Co. Ltd. (Dalian, China). The antibodies used were as follows: anti-tTG (ab109200, Abcam), anti-IL-33(ab54385, Abcam), Anti-ST2 (sc-74296, Santa Cruz Biotechnology, Dallas, USA), anti-COL I (14695-I-AP, Abcam, USA), anti-TLR4 (Ab47093, Abcam, USA), anti-p-p65 (sc136548, Santa Cruz Biotechnology), anti-p-65 (sc8008, Santa Cruz Biotechnology), anti-p-IKKα/β (ab178870, Abcam), anti-IKKγ (SC166700, Santa Cruz Biotechnology), anti-GAPDH (ab181603, Abcam), and HRP-conjugated secondary antibodies of mice or rabbit IgG (35552 and 35510, Invitrogen, Waltham, USA). Bicinchoninic acid (BCA) Protein Assay Kit was purchased from Guangzhou Dingguo Biotechnology (Guangzhou, China). Polyvinylidene fluoride membrane (PVDF) (ISEQ00010) was purchased from Merck Millipore (Darmstadt, Germany). 3,3′-diaminobenzidine (DAB) substrate kit was purchased from Gene Tech Company Limited (Shanghai, China). Cystamine (CTM), the inhibitor of tTG enzyme activity, TAK242, a TLR4 inhibitor were purchased from Sigma-Aldrich (St. Louis, USA) and Shanghai Haoyuan Chemoexpress (Shanghai, China), respectively.