Anti vegfr1

Protein samples were prepared from NRVMs using 1% sodium dodecyl sulfate (SDS) lysis buffer supplemented with protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche Diagnostics). Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), followed by blocking with 5% BSA (Sigma-Aldrich) in TBST (0.1% Tween-20 in Tris-buffered saline: 137 mM NaCl and 20 mM Tris-HCl, pH 7.4) for 1 h at room temperature. For NFAT, 8% skim milk (Difco, Detroit, MI, USA) in TBST was used for blocking. Membranes were then incubated overnight at 4°C with the following primary antibodies: anti-VEGFR1, anti-PKG-1, anti-phospho (p)-CaMKII (Thr286) (Cell Signaling Technology, MA, USA), anti-p-NFATc3, anti-p-NFATc4, anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Next, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (AbFrontier, Seoul, Korea) at room temperature for 1 h, and luminescence was detected using an ImageQuant LAS 4000 mini (GE Healthcare) and a SuperSignal West Pico Chemiluminescence Kit (Thermo Fisher Scientific, Waltham, MA, USA). The intensity of each protein band was quantified by NIH ImageJ software.