Quantitative Analysis of PDGFB Expression

Quantitative reverse transcription (qRT)-PCR was performed with the iScript reverse transcription and iTaq SYBR Green qPCR kit (BioRad, Hercules, CA) using the BioRad real time PCR system (BioRad). β2 microglobulin controlled for overall cDNA content as a reference gene. The primers used for human β2 microglobulin and Twist1 were previously described^{12 (link),13 (link),20 (link),34 (link)}. The primers used for human PDGFB were forward; 5′-CTCGATCCGCTCCTTTGATGA-3′ and reverse; 5′-CGTTGGTGCGGTCTATGAG-3′. The protein levels of human PDGFB in ECs were measured using ELISA (R&D systems) and normalized by the protein levels of total cell lysate. When we measured the PDGFB protein levels in the conditioned media (CM), we normalized the levels by the cell numbers.