Western blotting (WB) analysis
Total protein was extracted from spleen or aorta by High E ciency RIPA Lysis Buffer (KeyGEN BioTECH) and quanti ed by BCA protein concentration assay kit (Beyotime Biotechnology). Protein loading buffer (Beyotime Biotechnology) was added into protein and boiled for 5 minutes for protein degeneration. 30 µg of protein was loaded in 8% SDS-PAGE gel and transferred onto polyvinylidene uoride (PVDF) membranes (Merck Millipore). Skim milk in TBST (TBS containing 0.1% Tween 20) was used as a blocking agent before antibody incubation. PVDF membranes were incubated with rabbit antibodies against Dnmt3b (Cell Signaling Technology) and GAPDH (Abways), mice antibody Dnmt1 (Abcam). Goat anti-rabbit IgG and goat anti-mice with HRP (Proteintech) were chosen as the secondary antibody. The imaging was performed in Gel Doc XR Biorad (Bio-Rad) using Chemiluminescent HRP Substrate (Merck Millipore). Data was analyzed with Image Lab 4.0 (Bio-Rad). Gray values of blot areas were measured, and the relative expression amount of the protein samples was calculated by the method of the target protein gray value/internal reference GAPDH gray value.
Total protein was extracted from spleen or aorta by High E ciency RIPA Lysis Buffer (KeyGEN BioTECH) and quanti ed by BCA protein concentration assay kit (Beyotime Biotechnology). Protein loading buffer (Beyotime Biotechnology) was added into protein and boiled for 5 minutes for protein degeneration. 30 µg of protein was loaded in 8% SDS-PAGE gel and transferred onto polyvinylidene uoride (PVDF) membranes (Merck Millipore). Skim milk in TBST (TBS containing 0.1% Tween 20) was used as a blocking agent before antibody incubation. PVDF membranes were incubated with rabbit antibodies against Dnmt3b (Cell Signaling Technology) and GAPDH (Abways), mice antibody Dnmt1 (Abcam). Goat anti-rabbit IgG and goat anti-mice with HRP (Proteintech) were chosen as the secondary antibody. The imaging was performed in Gel Doc XR Biorad (Bio-Rad) using Chemiluminescent HRP Substrate (Merck Millipore). Data was analyzed with Image Lab 4.0 (Bio-Rad). Gray values of blot areas were measured, and the relative expression amount of the protein samples was calculated by the method of the target protein gray value/internal reference GAPDH gray value.