Sybr premix ex taq 2 rr820a

Total RNA, enriched for miRNAs and mRNA, was extracted using the miRNeasy Mini kit (Qiagen, Germany) and TransZol Up Plus RNA kit (TransGen, China) from whole insect bodies (n = 30), respectively. The miScript Ⅱ RT kit (QIAGEN) and the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen) were used to prepare cDNA. Quantitative real-time PCR (qPCR) reactions were conducted with an ABI Prism 7300 (Applied Biosystems, USA) using miScript SYBR Green (Qiagen, Germany) and SYBR® Premix Ex Taq II RR820A (Takara, Japan), according to the manufacturer’s instructions. The data was analyzed using the 2^-△△Ct method. Actin1 (GenBank accession No. EU179846.1) was used as the endogenous control. This gene has been studied to show it is suitable to be used as a reference gene [46 (link)]. All treatments were carried out in triplicate. Independent reactions were performed in quadruplicate for each RNA sample, and the signal intensity of the target gene is presented as the average value. The qPCR primers used in this study are listed in S3 Table.

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.