Anti phospho fak tyr 397 anti fak

We used immunoprecipitation assay of western blotting to determine the levels of phosphorylation of NMIIA. Protein G Immunoprecipitation Kit (IP-50, Sigma-Aldrich, MO, USA) was utilized to interact with erythrocyte lysate overnight. Antibodies used in this process were purchased from ab55456 (Abcam Ltd., Cambridge, UK) for NMIIA. We used anti-phosphotyrosine antibody, clone 4G10, purchased from 05 to 321 (Millipore, MA, USA), to measure the tyrosine phosphorylation level in immunoblots. Anti-phospho-VASP (Ser 239 )/anti-VASP (ab194747 and ab109321, Abcam Ltd., Cambridge, UK) and anti-phospho-FAK (Tyr 397 )/anti-FAK (#8556 and #3285, Cell Signaling Technology, MA, USA) were used as primary antibodies to detect the phosphorylated and total proteins of VASP and FAK. Specific antibodies for detecting total and phosphorylated eNOS proteins were used, including anti-phospho-eNOS (Ser 1177 )/anti-eNOS (GTX129058/GTX129113, GeneTex Inc., CA, USA) as primary antibodies. Antibodies for β-actin (sc-47778, Santa Cruz Biotechnology Inc., CA, USA) were diluted 1:20,000 in 3% bovine serum albumin. Specific bands by immunoblotting reaction were visualized using the enhanced chemiluminescence system (Millipore, MA, USA). The intensity of blot band was quantified through the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The level of AQP1 phosphorylation was detected by an immunoprecipitation assay. The method of AQP1 immunoprecipitation has been described in our previous article. [ 13 ] The level of Glut1 phosphorylation was also detected by immunoprecipitation assay. We used specific antibodies, anti-phospho-FAK (Tyr 397 )/anti-FAK (#8556 and #3285, Cell Signaling Technology, MA, USA), to detect total and phosphorylated FAK proteins (Tyr 397 ) and antibodies, Abcam and ThermoPierce (Abcam, Cambridge, UK; Thermo Fisher Scientific, MA, USA), to detect total and phosphorylated Glut1. Specific bands from an immunoblotting reaction were visualized using the Enhanced Chemiluminescence System (Millipore, MA, USA). The intensity of each band was analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).