H3k9me2

Specifically, for the Western blot of nuclear proteins, the nuclei of cells were isolated using Nucleoprotein Extraction Kit (Cat: C500009, Sangon Biotech, Shanghai, China). After quantified with Pierce™ BCA Protein Assay Kit (Cat: 23225, ThermoFisher Scientific, Waltham, MA USA), equal protein samples were resolved in 10% SDS-PAGE gel. Then, the separated proteins were transferred onto nitrocellulose filter (NC) membrane. After blocked in 5% non-fat milk/PBS for 1 h at room temperature, the membranes were washed with TBST for 3 × 5 min and then incubated with primary antibody at 4ºC overnight. The next day, the membranes were washed with TBST for 3 × 5 min at room temperature, and then were probed with the HRP (peroxidase)-conjugated secondary antibody at room temperature for 1 h. Signals were detected using Pierce ECL Western Blotting Substrate (Cat: 32106, ThermoFisher Scientific, Waltham, MA USA) on Tanon 5200 multi-automatic image analysis system (Tanon, Shanghai, China). The protein bands were quantified using ImageJ software. Primary antibodies of EHMT2 (Cat: #3306), β-Actin (Cat; #3700), β-catenin (Cat: #8480) were purchased from Cell Signaling Technology (Danvers, USA). H3K9me2 (Cat: 49-1007) was purchased from ThermoFisher Scientific (Waltham, MA USA).

Protein levels were quantified by Bradford assay. The protein sample was diluted, heated for denaturation, then subjected to dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore). The membrane was blocked in 0.1% Triton X-100 for nuclear proteins and Tween 20 for cytoplasmic proteins, and 5% non-fat milk powder in phosphate-buffered saline for 1 h at 4°C. Primary antibodies against H3K4me1 (ThermoFisher), H3K4me2 ThermoFisher), H3K9me1 (ThermoFisher), H3K9me2 (ThermoFisher), Histone H3 (ThermoFisher), KDM1A (ThermoFisher), Vimentin (ThermoFisher), E-cadherin (ThermoFisher), N-cadherin (ThermoFisher), GAPDH (Abcam), and β-actin (Abcam) were then added, and membranes were kept incubating at 4°C overnight. Corresponding secondary antibodies were applied followed by electrochemiluminescence processing.