Rabbit anti inos antibody

Protein expression level was determined by Western blotting technique with the aid of a panel of specific antibodies. Briefly, the peritoneal macrophages seeded in a 6-well plate were incubated with different concentrations of jacaric acid (either 50 or 100 μM) in the presence of LPS (60 ng/ml) at 37°C for 72 h. Cells treated with 0.1% ethanol acted as the control. After incubation, the cell pellet was collected and total protein was extracted by the cell lysis buffer. Protein concentration was measured by Bradford reagent and the protein samples (30 μg/well) were resolved on a 10% polyacrylamide gel and transferred to a PVDF membrane. The membrane was first incubated with the rabbit anti-iNOS antibody (diluted 1:250) (Cell Signaling Technology Inc., USA) or mouse anti-β-actin antibody (diluted 1:2,000) (Santa Cruz Biotechnology), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare Limited, UK) and finally developed with the enhanced chemiluminescence reagent (Santa Cruz Biotechnology).

Extracted proteins were quantified using a BCA Protein Assay Kit (Beyotime, Nantong, China). The protein extracts (50 μg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking with silk milk for 2 h at room temperature, the proteins were probed with primary antibodies and specific conjugated peroxidase-labeled secondary antibodies. The immunoreactive bands were visualized using a luminescent image analyzer (Amersham Imager 600; GE, MA, USA). Protein expression levels were determined by densitometry. All experiments were performed in triplicate. The primary antibodies used were as follows: rabbit anti-MCP-1 antibody (Cell Signaling Technology, MA, USA), rat ICAM-1 antibody (1 : 500; R&D Systems, MN, USA), rabbit anti-iNOS antibody (1 : 1000; Cell Signaling Technology), rabbit anti-LC3 antibody (1 : 1000; Cell Signaling Technology), rabbit anti-P62 antibody (1 : 1000; Cell Signaling Technology), and rabbit anti-GAPDH antibody (1 : 1000; Cell Signaling Technology).

INS‐1 cells were harvested with ice‐cold lysis buffer supplemented with complete proteinase inhibitor mixture (Roche). Other specific information of Western blotting assay was performed as previously described previously.¹⁹ The antibodies used are as follows: rabbit anti‐CASK antibody (1:1000; Cell Signaling), rabbit anti‐DNMT1 (1:1000; Cell Signaling), mouse anti‐DNMT3a (1:1000; Santa Cruz Biotechnology), rabbit anti‐DNMT3b (1:1000; Abcam), rabbit anti‐Akt(pan) antibody (1:1000; Cell Signaling), rabbit anti‐p‐Akt Ser473 antibody (1:5000; Abcam), rabbit anti‐iNOS Antibody (1:1000; Cell Signaling) or rabbit anti‐β‐tubulin antibody (1:4000; Cell Signaling).