Protein Separation and Detection by Western Blot

The total protein was separated by SDS-PAGE with 10% stacking gel and 12% separating gel at 80 V for 2 h and then transferred onto a PVDF membrane at 90 V for 55 min at 4 °C. Then, the membrane was blocked with 5% skimmed milk (diluted in TBS-T) at room temperature in a shaker for 1 h. After 3 washes with TBS-T, the membrane was incubated with the mouse GFP antibody [44 (link)] (1:4000, Proteintech Group, Wuhan, China), anti-GAPDH rabbit monoclonal antibody (1:30000, Boster, Wuhan, China) [45 (link)], or histone H3 antibody [46 (link)] (1:1000, Wanleibio, Shenyang, China) at room temperature for 2 h, followed by 3 washes with TBS-T. After that, the membrane was incubated with HRP-conjugated goat anti-mouse IgG(H+L) [47 (link)] (1:1000, Beyotime, Shanghai, China) or HRP-labeled goat anti-rabbit IgG(H+L) (1:1000, Beyotime, Shanghai, China) at room temperature for 1.5 h. Subsequently, the membrane was treated with ECL SuperSignal Solution [48 (link)] (Beyotime, Shanghai, China), and the protein bands were detected using a Bioanalytical Imaging System c600 (Azure Biosystems, Dublin, CA, USA).