Anti rabbit horseradish peroxidase conjugated secondary antibody

Twenty-five milligrams of detrusor tissue was ground on ice and added to Ripa lysate buffer containing phenylmethane sulfonylfluoride and a phosphatase inhibitor. The protein concentration was measured using a bicinchoninic acid protein detection kit (Beyotime Biotechnology, China). The protein samples were separated by electrophoresis in 10% twelve alkyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride film. The membrane was sealed in Tris-Buffered Saline Tween-20 with 5% skim milk for 1 h and incubated with the primary antibody at 4°C overnight. The solution was replaced the next day with horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:5000) (Boster Biological Technology, China) and incubated at room temperature for 1 h. After washing, the band was visualized with a super-sensitive electrogenerated chemiluminescence (ECL) chemiluminescence solution (Haigene Detection Co. Ltd, China) and analyzed with a Chemidoc MP chemiluminescence imaging system (Bio-Rad, USA). Actin was used as the internal reference and the following proteins were examined: Actin (Abcam, UK), SphK1 (Abcam), SphK2 (Thermo, USA), S1PR1 (Abcam), S1PR2 (Thermo), S1PR3 (Abcam), RhoA (Abcam), ROCK1 (Cst, USA), ROCK2 (Cst), MYPT1 (Cst), p-MYPT1 (Cst), MLC20 (Cst), and p-MLC20 (Cst).

Astrocytes and brain microvascular endothelial cells were collected after mimicking DD conditions, and radioimmunoprecipitation assay buffer containing phenylmethyl sulfonylfluoride was added to extract total protein. Proteins were then electrophoretically resolved on 10% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. The blots were blocked with skimmed milk and incubated with rabbit anti-occludin (1:1000; Cell Signaling Technology, Boston, MA, USA), rabbit anti-GFAP (1:1000; Cell Signaling Technology), rabbit anti-CX43 (1:1000; Cell Signaling Technology) or rabbit anti-GAPDH (1:1000; Cell Signaling Technology) antibody overnight at 4°C. The blots were washed twice with Tris-buffered saline containing Tween 20, and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Boster). Finally, the signals were visualized with an enhanced chemiluminescence kit (Amersham, Shanghai, China).