Rabbit anti h2ak119ub

HEK293T cells were lysed in 1× lysis buffer on ice for 30 min and centrifuged at 12,000 rpm for 15 min. Cell lysates were separated by electrophoresis on 4 to 20% SDS–polyacrylamide gel electrophoresis gels and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% milk in TBST (Tris-buffered saline, 0.1% Tween® 20) for 1 hour and incubated overnight at 4°C with the corresponding primary antibodies: rabbit anti-FLAG and anti–β-tubulin (Sigma-Aldrich), rabbit anti-H3K9me1 (Abcam), and rabbit anti-H2AK119ub (Cell Signaling Technology). Next, the experiment was followed by incubation with the horseradish peroxidase–labeled Goat Anti-Rabbit immunoglobulin G (Beyotime) for 1 hour at room temperature 20° to 25°C. Quantitation of immunoblots was then performed via densitometric analysis using the ImageJ software.