Supersignal west pico enhanced chemiluminescence kit

Mouse brains were minced and lysed in Pierce RIPA buffer (Thermo Fisher Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Tissue lysate was collected by centrifugation and quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). An equal amount of lysate was boiled in loading buffer and subjected to SDS-PAGE. Subsequently, the separated protein in gel was transferred onto the PVDF membrane, which was then blocked in 5% milk and incubated with anti-nectin1 mouse monoclonal antibody (sc-21722, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight. The membrane was washed and further incubated with HRP-conjugated goat-mouse IgG secondary antibody. After thorough washing, the target protein was imaged using a SuperSignal West Pico enhanced chemiluminescence kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The same blots were probed with a β-actin rabbit monoclonal antibody (13E5, Cell Signaling Technology, Danvers, MA, USA) as a loading control.