Anti immunoglobulin g horseradish peroxidase conjugate

To investigate protein expression of GPBAR1 and CSE, LSEC were serum starved overnight and then stimulated with 10 μM BAR501 for 18 h. In another experimental setting serum starved LSEC were incubated 5, 15, 30 and 60 minutes with 10 μM BAR501. Total lysates were prepared by solubilization of endothelial cells in E1A lysis buffer containing protease and phosphatase inhibitors. The proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad) and probed with primary antibodies CSE (Santa Cruz), GPBAR1/TGR5 (Abcam), tubulin (Sigma), phospho-Akt (Thr308—Santa Cruz), Akt (Santa Cruz), phospho-Serine (Abcam), phosphoeNOS (ser1177 –Cell Signaling), eNOS (Cell Signaling), phosphoFOXO1 (Thr24 –Santa Cruz) and FOXO1 (Santa Cruz). nitrocellulose membranes from immunoprecipitation (IP) experiments were first probed with a phospho-Serine antibody, stripped and then re-probed with the CSE antibody. Similarly, nitrocellulose membranes from IP experiments were first probed with the phospho-Akt antibody, stripped and then re-probed with the Akt antibody. The anti-immunoglobulin G horseradish peroxidase conjugate (Bio-Rad) was used as the secondary antibody, and specific protein bands were visualized using Super Signal West Dura (Pierce), following the manufacturer’s suggested protocol.

Pancreatic protein was extracted by nuclear and cytoplasmic extraction reagents according to the manufacture's instructions (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was determined using a commercial BCA protein assay kit (Pierce, Rockford, IL). For the immunoblotting analysis, proteins were separated by a 4% to 8% polyacrylamide gel and transferred by electrophoresis to polyvinylidene difluoride membranes (Millipore, Bedford, MA). For nonspecific bindings, membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween 20 overnight at 4°C. Then, the membranes were incubated with a diluted solution of anti-HMGB1 antibody (1∶200, Santa Cruz, CA) or anti-GAPDH antibody (1∶1000, Abcam, Cambridge, MA) at 4°C overnight. After incubation with the secondary antibody, anti-immunoglobulin G horseradish peroxidase conjugate (Bio-Rad, Hercules, CA), the membrane was exposed to a chemiluminescent reagent (Amersham Biotechnology Pharmacia, Piscataway, NJ). Specific protein bands were photographed, The band concentration was calculated by the quantification of the integrated optical density of the appropriate band using Quantity One software (Bio-Rad, Hercules, CA).