Quantification of SMURF2 Expression

Total RNA was extracted by Quick-RNA^TM MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA). Then 1 μg of extracted RNA was used for cDNA synthesis with random hexamers provided in RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) followed by SYBR Green-based qPCR reaction (SYBR^® FAST qPCR Kit, Kapa Biosystems, Inc., Wilmington, MA, USA). The cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 sec and 60 °C for 1 min. The end-point used in the real-time quantification was calculated by the StepOne software (v2.2.2, Applied Biosystmes, Carlsbad, CA, USA, 2011), and the threshold cycle number (Ct value) for each analyzed sample was calculated. The primer sets used in this study were listed as followed:

SMURF2

Forward: 5’-TAGCCCTGGCAGACCTCTTA-3’

Reverse: 5’- AATACACCTGGCCTTGTTGC-3’

MRPL19 (internal control)

Forward: 5’- GGGATTTGCATTCAGAGATCAG-3’

Reverse: 5’- GGAAGGGCATCTCGTAAG-3’

qPCR data were analyzed as previous described [35 (link)].