Primescript rt master mix perfect real time reagent

Neferine (CAS 2292-16-2, purity ≥98%) was purchased from Nature Standard Biological Technology Co., Ltd. (Shanghai, China). The NE-PER^® Nuclear and Cytoplasmic Extraction Reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Monoclonal antibodies of p38 MAPK, JNK, ERK 1/2, NF-κBp65, IκB, histone H3, and β-tubulin were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase- (HRP-) labeled glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody and goat anti-rabbit immunoglobulin G (IgG) secondary antibody were provided by Bioworld Technology, Inc. (St Louis Park, MN, USA) and Signalway Antibody, Inc. (Park MD, USA), respectively. The SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) reagent and the PrimeScript™ RT Master Mix (Perfect Real Time) reagent were purchased from TaKaRa Co., Ltd. (Kyoto, Japan). The diaminobenzidine colorimetric kits and rabbit IgG immunohistochemistry (streptavidin-biotin complex) were purchased from BOSTER Biological Technology Ltd. (Wuhan, China).

We performed experiments with reference to previous studies [33 (link)]. AGS GC cells were obtained from Zhongqiao New Prefecture in Shanghai. All cells were grown in medium containing 10% fetal bovine serum (FBS; Gibco, NY, USA) and 1% penicillin–streptomycin (HyClone, Logan, UT, USA) in a standard humidified incubator. Total RNA was extracted using TRIzol Reagent (TaKaRa, Beijing, China) and reverse transcribed into cDNA using the PrimeScript RTMaster Mix (Perfect Real Time) reagent (TaKaRa, Beijing, China) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7500HT Fast Real-Time PCR System (Applied Biosystems, CA, USA). The average fold of relative mRNA expression was determined using the 2^−ΔΔCt method with GAPDH as an internal control [33 (link)]. Primer sequences for qRT-PCR were as follows:

ST6GALNAC3, forward 5′-ACCAGCGTTCCTCTTTTGCT-3′ and reverse 5′-TCATGCGCTTCTCTGTGGTC-3′;

ROR2, forward 5′-AAGGAACCTCCCCAGCCA-3′ and reverse 5′-GCCACCACCCCTTTCTACG-3′;

TAGLN, forward 5′-CCATGCCAGACAGCAGAGG-3′ and reverse 5′-ACTCTGCTTTGGAGTACAGCC-3′;

GAPDH, forward 5′-TCGACAGTCAGCCGCATCTT-3′ and reverse 5′-GAGTTAAAAGCAGCCCTGGTG-3′.