Western blot analyses

VSMCs irradiated with FIR radiation in the absence or presence of various chemicals were lysed in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 mM β-glycerophosphate, 1 mM NaF, 1 mM Na₃VO₄, and 1× protease inhibitor cocktail from Roche Molecular Biochemicals [Indianapolis, IN, USA]). In addition to VSMCs, the endothelium-deprived rat aortas were either irradiated with FIR radiation or not. The proteins were extracted by chopping the aortic tissues using iris scissors in an ice-cold lysis buffer as previously described.28 Protein concentration was determined using a BCA protein assay kit from Thermo Scientific (Rockford, IL, USA). Equal quantities of protein (20 µg) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane from GE Healthcare Life Sciences (Pittsburgh, PA, USA). The blots were probed with the appropriate primary antibodies, followed by the corresponding secondary antibodies, all from Invitrogen (Carlsbad, CA, USA), and developed using ECL reagents from Amersham Biosciences (Arlington Heights, IL, USA). The primary antibody dilutions used for western blot analyses were as follows: p-mTOR-Ser²⁴⁴⁸ (1:1,000), mTOR (1:1,000), p-p70S6K-Thr³⁸⁹ (1:1,000), p70S6K (1:1,000), p-AMPK-Thr³⁸⁹ (1:1,000), AMPK (1:1,000), and GAPDH (1:3,000).