Ecl chemiluminescent detection kit

Total protein (500 μg) was incubated with anti-CRMP2 (1:50; Abcam, ab129082) or anti-α-tubulin (1:50) (Abcam, ab7291) overnight at 4°C. An equal amount of protein was incubated with mouse IgG (Beyotime, A7028) or rabbit IgG (Beyotime, A7016) as negative controls. The protein samples were loaded as the input control. Protein A + G agarose (Beyotime, P2012) was washed with PBS three times and mixed with the samples. The mixtures were incubated at 4°C for 2 h. The beads were then washed with PBS three times. The immunoprecipitates were subsequently subjected to 12% SDS-PAGE and analyzed via western blot using anti-α-tubulin (1:5,000) or anti-CRMP2 (1:20,000) antibodies. The protein bands were visualized using an ECL chemiluminescent detection kit (Beyotime, P0018S).

The proteins from the hippocampus were separated using a 10-12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk for 1 h at room temperature and incubated with primary antibodies overnight at 4°C; the primary antibodies included anti-CRMP2 (1:20,000; Abcam, ab129082); anti-pCRMP2 (Thr514 site) (7 μl: 5 ml; Abcam, ab85934); anti-DNMT1 (1:5,000); anti-DNMT3a (1:5,000); anti-DNMT3b (1:5,000); anti-α-tubulin (1:5,000; Abcam, ab7291); anti-Tyr-Tubulin (1:2,500; Sigma, T9028); anti-Acet-Tubulin (1:200; Santa Cruz Biochemistry, sc-23950); and anti-GAPDH (1:5,000; Beyotime, AF1186). After washing three times, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Beyotime, 1:5,000) at room temperature for 1 h. Then, the membranes were visualized by an ECL chemiluminescent detection kit (Beyotime, P0018S), and the intensities of the protein bands were calculated by ImageJ software. The relative expression of the proteins was normalized to that of GAPDH.