Horseradish peroxidase conjugated goat anti rabbit or goat anti mouse secondary antibody

Western blot analysis was used to quantify the temporal changes in protein levels, and three mice per group were used. Briefly, brains were quickly removed from deeply anesthetized male WT and TG mice and stored at −80°C. After homogenization in lysis buffer containing protease inhibitor and phenylmethylsulfonyl fluoride, the protein concentrations of each group were assessed using BCA assays. The total protein was separated by SDS–PAGE (8%) with 3-mg protein in each lane and transferred to PVDF membranes. After blocking in 10% nonfat dry milk for 2 h at RT, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:5000, Santa Cruz Biotechnology) for 2 h at RT. Secondary antibody reactive bands were visualized with chemiluminescence reagents (ECL, Pierce) for 1 min and exposed onto X-films for 2–10 min. The band intensity was quantified using Image software and normalized to GAPDH internal controls.

Equal amounts of proteins were separated by 10% SDS-PAGE gels and blotted onto nitrocellulose membranes. The blots were incubated with primary antibodies against ARHGAP5 (1:1000; Abcam, Cambridge, USA) and β-Actin (1:5000; Santa Cruz Biotech, Santa Cruz, CA, USA) at 4°C overnight. The membranes were incubated for 2 h with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (1:5000; Santa Cruz Biotechnology, CA, USA) for 1 h at room temperature. Anti-β-Actin antibody (Santa Cruz Biotechnology, CA, USA) was used as a loading control.