Horseradish peroxidase labeled antibody

Aquaporin 4 protein expression level was detected by Western blot. After MRI examination, rats were rapidly perfused intracardially with cold PBS and then decapitated quickly. The Infarcted cerebral hemisphere was isolated on ice and homogenized. The concentration of protein in the resulting supernatants was calculated using a BCA protein assay kit. Samples containing approximately 40 μg of protein were loaded, separated using 10% SDS-polyacrylamide gel electrophoresis, and then transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA, USA). After blocking with Tris–buffered saline containing 5% skimmed milk for 2 h, the blot was then incubated overnight at 4°C with primary antibodies, including AQP4 (1:1300, Santa Cruz Biotechnology, TX, USA, SC-20812), β-actin (1:1000, Beyotime, Shanghai, China, AA128). The blot was subsequently incubated with horse radish peroxidase-labeled antibody (1:1000, Beyotime, Shanghai, China, A0208) for 1 h at RT. The membrane was then incubated in ECL solution, and the specific bands were captured and quantified with a ChemiDocMP (Bio-Rad, CA, USA). The AQP4 protein levels were normalized to β-actin.

To evaluate the activation of INSR and its downstream signaling, total INSR, phospho-INSR and total Akt, phospho-Akt were measured by western blot with and without insulin stimulation. A Flag-tagged INSR expression vector (wild or mutant type) was transfected into CHO cells. For phosphorylation assays, CHO cells were serum-starved 48 h after transfection for 4 h, before stimulation with 10 nmol/L insulin or vehicle control for 5 min at 37°C. 40 μg of total protein was resolved by 10% SDS-PAGE before transfer to PVDF membranes. The membranes were then incubated with anti-Flag (1:3000, Sigma-Aldrich), anti-INSR (1:1000, Abcam), anti-phosphorylated INSR (1:1000, Abcam), anti-Akt (1:1000, Cell Signaling Technology), anti-phosphorylated Akt (1:1000, Cell Signaling Technology), or GAPDH (1:3000, ProTech), followed by secondary horseradish peroxidase-labeled antibody (1:3000, Beyotime, China). Membranes were visualized by chemiluminescence reaction. Band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD).

Radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors was used to lyse the NR8383 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) was used to separate the proteins from 20 μg of cell lysate, and then the proteins were transferred to polyvinylidene fluoride membranes. Primary antibodies against NLRP3, ASC, P10, SIRT1 and β-actin (Beyotime, Wuhan, China) were used to blot the membranes, and then the corresponding secondary antibodies were applied (horseradish peroxidase-labeled antibody; Beyotime). Pierce^TM ECL Plus Western blot substrate was used to visualize the immunoblots.