Phosphorylated p38

As described previously [16] , protein extracted from lung tissues and human pulmonary arterial smooth cells (PASMCs) were separated via 10% SDS-PAGE, electroblotted to a PVDF membrane, and incubated with primary antibodies against DPP-4 (Abcam), α-SMA (Abcam), von Willebrand factor (vWF, Proteintech, Rosemont, IL), cas-pase3 (CST), phosphatase, and tensin homolog deleted on chromosome ten (PTEN, CST), extracellular regulated protein kinases 1/2 (ERK1/2, CST), phosphorylated ERK1/ 2 (p-ERK1/2, CST), protein kinase B (AKT, CST), phosphorylated AKT (p-AKT, CST), c-Jun N-terminal kinase (JNK, CST), phosphorylated JNK (p-JNK, CST), mitogenactivated protein kinase (MAPK) p38 (CST), phosphorylated p38 (CST), and β-actin (Proteintech). Over 16 h later, PVDF membrane was incubated with HRP-conjugated second antibody and chemiluminescence (ECL, CST) were employed to label protein band. Band intensities were analyzed densitometrically using Image Lab 4.1 software.
RNA derived from lung tissues and human PASMCs was extracted using Trizol reagent (Gibco BRL, Grand Island, NY). Reverse transcription was conducted with 2000 ng of total RNA with SYBR Premix Ex Taq™ kit (TaKaRa Bio Inc., Dalian, China). Quantitative RCR was performed using an ABI Prism 7500 FAST apparatus (Applied Biosystems, Foster City, CA, USA). β-actin was selected as housekeeping gene. PCR primers are listed in Table 1.