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ELISA, Western blotting, and Quantitative RT-PCR assay

DY Duomeng Yang
XD Xiaomeng Dai
YX Yun Xing
XT Xiangxu Tang
GY Guang Yang
AH Andrew G. Harrison
JC Jason Cahoon
HL Hongmei Li
XL Xiuxiu Lv
XY Xiaohui Yu
PW Penghua Wang
HW Huadong Wang
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NE and TNF-α concentration in heart tissues, perfusate, and cell supernatants were determined using the NE research enzyme-linked immunosorbent assay (ELISA) kit (ALPCO#17-NORHU-E01-RES) and the TNF-α Quantikine ELISA kit (R&D System#RTA00), respectively. The western blotting assays of proteins were performed using standard protocol22. Antibodies for western blotting assays are available in the Supplementary Methods. Quantitative RT-PCR assay of relative mRNA expression was analyzed using standard real-time PCR protocol according to TAKARA qPCR kits. In brief, total RNA was reverse transcribed using a PrimeScriptTM RT Reagent Kit (TAKARA#RR047A). Real-time PCR were performed with the SYBR Premix Ex Taq II (TAKARA#RR820A) in a LightCycler480 real-time PCR system. Primer sequences for genes are shown in Table S3.

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