Phospho enos ser1177 p enos

Western blotting was carried out as previously described [10 (link)]. Briefly, rat muscle and kidney tissue were homogenized and centrifuged, and proteins in the lysate were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Proteins were detected on membranes using antibodies against eNOS (Cell Signaling Technology, Tokyo, Japan), phospho-eNOS (Ser1177) (p-eNOS; Cell Signaling Technology), inducible NO synthase (iNOS; Cell Signaling Technology), heme oxygenase (HO)-1 (Thermo Fisher Scientific K.K.), α-tubulin (Cell Signaling Technology), and hypoxia-inducible factor-1 alpha (HIF-1α) (Abcam PLC, Tokyo, Japan). Protein bands were visualized using an enhanced chemiluminescence detection system (SuperSignal West Dura Extended Duration Substrate; Pierce Biotechnology, Tokyo, Japan) with horseradish peroxidase-conjugated secondary antibodies (Pierce Biotechnology). Band intensities were quantified using a ChemiDoc XRS + Molecular Imager with Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA), with α-tubulin used as a loading control.

Western blot technique was used for protein analysis of cell lysates as previously described [12 (link)]. Briefly, sample media was mixed with 4X loading (sample) buffer (Sigma, St Louis, MO) and Radio-Immuno Precipitation Assay buffer, pH 7.4 (Cat No. BP-115, Boston BioProducts, Ashland, MA). 15–50 μg of sample was subjected to Western blot depending on target proteins. The same amount of protein was used within a Western blot and target proteins were then visualized with an enhanced chemiluminescence detection system. Antibodies used in this study were for endothelial nitric oxide synthase (eNOS) (Cat No. 9572; Cell signaling technology, Danvers, MA) 1:1000; phospho-eNOS (Ser1177) (p-eNOS) (Cat No. 9571; Cell signaling technology, Danvers, MA) 1:1000; α-Klotho (Cat No. Ab75023; Abcam, Cambridge, MA) 1:1000; phospho-FGFR (Tyr653/654) (p-FGFR) (Cat No.3471; Cell signaling technology, Danvers, MA) 1:1000; FGFR1 (Cat No. SC-121; Santa Cruz Biotechnology, Santa Cruz, CA) 1:1000; FGFR3 (Cat No. SC-123; Santa Cruz Biotechnology, Santa Cruz, CA) 1:1000; FGFR4 (Cat No. Ab5481; Abcam, Cambridge, MA) 1:1000 and Actin (Cat No. MAB1501; Millipore, Billerica, MA) 1:1000.