Rabbit anti nf κb p65

A cell scraper was used to quickly collect cells from each group, and nuclear and cytoplasmic proteins were extracted according to the instructions of a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China, P0028). The protein concentrations were determined by the BCA method. Thirty micrograms of protein was added to a 10% SDS-PAGE gel and then transferred to polyvinylidene uoride membranes. The membranes were incubated with fast blocking solution and then incubated with the following primary antibodies overnight at 4°C: rabbit anti-NF-κB p65 (1:2000, Beyotime, Shanghai, China, AF0246), rabbit anti-ErbB4 (1:500, Abcam, United Kingdom, ab32375), rabbit anti-p-ErbB4 (1:500, Abcam, United Kingdom, ab109273), mouse anti-Bax (1:1000, SANTA, Shanghai, China sc-7480), mouse anti-NRG1 (1:1000, SANTA, Shanghai, China, sc-393006), rabbit anti-histone H3
(1:1000, Beyotime, Shanghai, China, AF5614), rabbit anti-GAPDH (1:5000, Abcam, United Kingdom, AF1186), and rabbit anti-β-actin (1:5000, Proteintech, Chicago, USA, 66009-1-lg). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL, USA) for 2 h at room temperature. The bands were detected by enhanced chemiluminescence (ECL). ImageJ software was used to assess the optical density values.

BV2 cells and SH-SY5Y cells were detached with trypsin and seeded in a 24-well culture plate at a density of 10 4 cells/well. Polylysine-coated glass slides were placed in the bottom of the 24-well plate in advance. The cells were xed with 4% paraformaldehyde for 20 min at room temperature and washed 3 times with PBS. Triton X-100 (0.1%) was used to permeabilize the cell membrane for 20 min at room temperature. The cells were incubated with rabbit anti-NF-κB p65 (1:400, Beyotime, Shanghai, China, AF0246) and mouse anti-Bax (1:100, SANTA, Shanghai, China sc-7480) primary antibodies overnight at 4°C, incubated with secondary antibody (Beyotime, Shanghai, China, A0216, A0208) and 4', 6-diamidino-2-phenylindole (DAPI, Beyotime, Shanghai, China, C1006) for 1 h at room temperature.