Rt reaction kit

A reverse transcription reaction kit was purchased from Takara Biotechnology Co. Ltd. (Dalian, China); TRIzol was from Invitrogen Life Technologies (Carlsbad, CA, USA) and electrophoresis reagents were from Promag Co. (Ningbo, China); an RT Reaction kit was obtained from Takara Biotechnology Co. Ltd (Dailan, China); a PCR Amplification Reagent kit and the 100 bp DNA ladder marker were obtained from Sangon Biological Engineering Co. Ltd. (Shanghai, China); GAPDH was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-mouse NF-κB and VEGF polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China); M. suaveolens Extract Tablets were from Seiko Eiyo Yakuhin Co. Ltd. (Osaka, Japan); fluorescein isothiocyanate (FITC)-albumin and hexadecyl-trimethyl-ammonium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA) and SYBR green I was obtained from Biotium (Hayward, CA, USA). The Oligo (dT18) and primers were synthesized by Shanghai Invitrogen (Shanghai, China). The dNTP was obtained from Promega Corp. (Madison, WI, USA).

Total RNA was purified from cells in 60-mm dishes using TRIzol (Invitrogen) according to the manufacturer’s instructions. Then, cDNA was obtained from total RNA extracted from cells using a reverse transcription (RT) reaction kit (TaKaRa, Japan). The cDNA level was determined by SYBR Premix Ex Taq™Ⅱ (TaKara, Japan), and the procedure was carried out as follows: 95°C for 30 s for one cycle, 95°C for 5 s, and 60°C for 30 s, followed by plate reading for 40 cycles. The PCR primers (Supplementary Table S1) were designed using Primer3 plus. The relative expression levels of the mRNAs in the groups were analyzed using the 2ΔΔCT method.

Total RNA was purified from cells in 60-mm dishes using TRIzol (Invitrogen) according to the manufacturer’s instructions [35 (link)]. Then, cDNA was obtained from total RNA extracted from cells using a reverse transcription (RT) reaction kit (TAKARA, Japan) [13 (link), 36 (link)]. cDNA was completed with SYBR Premix Ex Taq™II (TaKaRa, Japan), and the program was carried out as follows: 95 °C for 30 s for one cycle; then, 95 °C for 5 s and 60 °C for 30 s, followed by plate reading for 40 cycles. The PCR primers (Table S1) were designed using Primer3 plus in Supplementary Table 1. The relative expression levels of the mRNAs in the groups were analyzed using the 2ΔΔCT method [32 (link), 37 (link)].

A reverse transcription reaction kit was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). TRIzol and electrophoresis reagents were from ProMag Co., Ltd. (Ningbo, China). The RT reaction kit was obtained from Takara Biotechnology Co., Ltd. The PCR amplification reagent kit and DNA ladder/marker were obtained from Shanghai Sangon Biological Engineering Co., Ltd. (Shanghai, China). GAPDH was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-mouse Tollip, TLR4, TRAF6, p-IRAK1, VEGF-α, HO-1 and NF-κB polyclonal antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Rabbit anti-mouse IRAK1 and phosphorylated IRAK1 were purchased from Cell Signaling, Technology (Beverly, MA, USA). FITC-albumin, hexadecyl-trimethyl-ammonium bromide were purchased from Sigma-Aldrich (St. Louis, MO, USA). SYBR-Green I was obtained from Biotium (Hayward, CA, USA). The Oligo(dT)18 and primers were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd (Shanghai, China). The dNTP was obtained from Promega (Madison, WI, USA).