Bcip nbt kit

The thoracic aortas were excised and homogenized in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS). After centrifugation at 12,000 g for 10 minutes, the resulting supernatant was processed to yield total protein samples. The protein concentrations were determined by the BCA Protein Assay Kit (Beyotime institute of Biotechnology, China) according to the manufacturer's instructions. Equal amounts of protein samples were boiled for 10 minutes in SDS-PAGE Sample Loading Buffer (Beyotime institute of Biotechnology, China) and loaded onto a 10% SDS-polyacrylamide gel before running. Then the proteins were transferred from the gel to 0.2 μm polyvinylidene fluoride (PVDF) membrane and blocked at room temperature for 2 hours. The membrane was incubated at 4°C overnight with primary antibodies including anti-VCAM-1 (1:200, Santa Cruz Biotechnology) and anti-inhibitory κBα (I-κBα, 1:2000, Abcam) followed by secondary antibody conjugated to alkaline phosphatase (1:1000, Zhongshan Golden Bridge Biotechnology Co., Beijing, China) at room temperature for 2 hours. Blots were developed using a BCIP-NBT kit (Promega, Madison, WI, USA). All blots were stripped and reprobed with polyclonal anti-β-actin to verify equal protein loading.

The total or nuclear proteins (n = 3) were extracted using commercially available kits according to the manufacturer’s protocol. Protein concentration was determined using the BCA protein assay kit (Beyotime institute of Biotechnology, China). Protein samples were analyzed using SDS-polyacrylamide gel electrophoresis (PAGE) followed by semi-dry transfer onto a PVDF membrane. The blots were then blocked with 5% nonfat milk and incubated with the appropriate primary antibodies, followed by incubation with a secondary antibody conjugated to alkaline phosphatase (Zhongshan Golden Bridge Biotechnology Co., Beijing, China). Immunoreactive bands were detected using a BCIP-NBT kit (Promega, Madison, WI, USA). The primary antibodies used were anti-VCAM-1 from Santa Cruz Biotechnology, Inc. (CA, USA), anti-MCP-1 from Boster Biological Technology, Ltd. (Wuhan, China), and anti-NF-κB, anti-inhibitory κBα, anti-p-JNK and anti-p-p38 MAPK from Abcam (Cambridge, UK).