Quantitative Analysis of Smurf2 and ALK5 mRNA

Total RNA was extracted with the TRIzol reagent (16096020, Thermo Fisher Scientific), and the cDNA of mRNA was synthesized using the first-strand synthesis kit (D7168L, Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the instructions. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was carried out using a RT-qPCR kit (Q511-02, Vazyme Biotech, Nanjing, China) according to the instructions. PCR amplification was carried out with Bio-rad real-time quantitative PCR instrument CFX96. β-actin served as the internal reference for Smurf2 and ALK5. All primer sequences were designed and provided by Sangon Biotech. The primer sequences are shown in Table 1. The relative mRNA expression was measured using the 2-^ΔΔCT method (Zhao et al., 2018 (link); Wan et al., 2019 (link)).