T enos

Western blotting was performed according to standard protocols described previously (Citation15). Briefly, thoracic aortas and CD4 + T cells were homogenized using icecold RIPA lysis buffer. The proteins lysates were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked using 8% fat-free milk in TBS (Tris-buffered saline) with 0.5% Tween-20 for 2 h at room temperature and then probed with primary antibodies against phosphorylated eNOS at Ser 1177 (1:500; Cell Signaling Technology, Danvers, MA, USA), t-eNOS (1:1000; Cell Signaling Technology), p-STAT3 (1:400, Abcam), t-STAT3 (1:400, Abcam), ROR-γt (1:400, Abcam) and GAPDH (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies at 4°C overnight. The anti-rabbit secondary antibody (1:10000, Li-Cor Bioscience, Bad Homburg, Germany) was incubated for 2 h at room temperature. The protein bands were detected by Odyssey Western Blot Detection System (LI-COR) and the band intensities were evaluated with ImageJ.