Total rna extraction reagent

Total RNA was extracted from RAW264.7 cells using the RNAiso Plus kit. Total RNA extraction reagent (Takara, Shiga, Japan) was used and cDNA was synthesized using the BioFact RT Series kit. PCR amplification was monitored by CFX Connect Real-Time PCR software (version 1.4.1; Bio-Rad Laboratories, Hercules, CA, USA). The primers used are listed in Table 1.

Total RNA was extracted from each sample by using a total RNA extraction reagent (Takara, Kusatsu, Japan), and was then reverse transcribed into cDNA with Mir-X miRNA First-Strand Synthesis kit (Takara, Kusatsu, Japan). The sequences of the miRNA-specific primers for real-time qPCR are listed in Table 2, and the reverse primer was the mRQ 3′ primer provided in Mir-X miRNA First-Strand Synthesis kit. qRT-PCR was performed using SYBR Premix Ex Taq (Takara, Kusatsu, Japan) for selected miRNAs. The reaction volume was 25 μL, and the qRT-PCR conditions were as follows: 30 s at 95°C, 40 cycles of 5 s at 95°C, and 30 s at 60°C, followed by a melting curve analysis step. Every sample was repeated three times, and relative quantification was performed by the 2^−ΔΔCq method. U6 was used as the endogenous control to normalize the data.

Total RNA was extracted and purified from the cells using the RNA isolator Total RNA Extraction Reagent (TaKaRa, Kusatsu, Japan) and subjected to reverse transcription using the PrimeScript™ RT Master Mix Kit (TaKaRa, Kusatsu, Japan). The real-time PCR and data collection were subsequently performed as described previously [30 (link)] using the AceQ® qPCR SYBR® Green Master Mix kit (TaKaRa, Kusatsu, Japan) on ABI 7500 system (ABI, New York, USA). Primers used for the amplification were as follows: TRIM72-F: 5′-CGAGCAGGACCGCACACTT-3′, TRIM72-R: 5′-CCAGGAACATCCGCATCTT-3′; insulin receptor substrate-1 (IRS-1) F: 5′-GAAGAAGTGGCGGCACAAGT3′, IRS-1 R: 5′-GTCAGGCAGAGGCGGTAGAT-3′; IRS-2 F: 5′-ATACCGCCTATGCCTGTCTG-3′, IRS-2 R: 5′-AGAAGAAGCTGTCCGAGTGG-3′; IRS-3 F: 5′-GCAGAGCAGCAAACATGGTA-3′, IRS-3 R: 5′-GCGAAGATCCAAGACTCAGG-3′; IRS-4 F: 5′-TTGCTGACAGTGCCATTTGC-3′, IRS-4 R: 5′-TGCACTTCTTCCTGCCTAGC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) F: 5′-GC ACCGTCAAGGCTGAGAAC-3′, GAPDH R: 5′-TGGTGAAGACGCCAGTGG A-3′. The relative expression levels of the indicated mRNA normalized against GAPDH mRNA were calculated using the 2^−ΔΔCT methods.

Total RNA extraction reagent (TaKaRa, Dalian, China) was used to extract the total RNA from the RVLM. The mRNAs of prorenin, PRR, NLRP3, pro-Casp-1, ASC, IL-1β, TNF-α, IL-10, and TGF-β were analyzed by quantitative real-time PCR. Isolated mRNA was quantified by spectrophotometry and the 260/280 nm optical density ratio was calculated. The cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems, ABI). The relative quantification of gene expression was expressed as fold-change via normalization against β-actin by using the 2ΔΔCT method. The sequences of primers were designed using Primer Express 2.0 and are listed in Table S1.

Chemicals and reagents used in this study were purchased from the following sources: corn oil and tamoxifen from Aladdin (Shanghai, China); Taq Plus Master Mix Ⅱ, GelRed Nucleic Acid Stain, agarose gel, DNA markers, and DNA Isolation Kit from Vazyme (Nanjing, China); total RNA extraction reagent, RT-PCR kit, and SYBR Premix Ex Taq™ kit from Takara (Dalian, China); eIF3a and GAPDH antibody from Abcam (Cambridge, UK); methanol, ethanol, acetone, and acetonitrile from Sinopharm (Shanghai, China); and BCA Protein Assay Kit from Invitrogen (Grand Island, United States).