Rabbit anti h3k9me1

Tissue was homogenized in lysis buffer (RIPA buffer, DTT; Cell Signaling, Danvers, MA, USA). Supernatant containing proteins was collected after 10 min centrifugation (10 000 g at 4°C). Protein concentration was assessed using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rochester, NY, USA). Protein samples were denaturated at 70 °C for 10 min and run on a 4–12% Bis-Tris gel (NuPAGE Novex; Life Technologies, Grand Island, NY, USA), and then transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% nonfat dry milk and incubated overnight with the primary antibody (rabbit anti-H3K9me1 (1/1000; Abcam) and rabbit anti-H3 (1/1000; Cell Signaling). The membrane was washed with TBST and then incubated with secondary antibody anti-rabbit HRP (1:10 000; Cell Signaling) for 1 h. Detection and densitometric evaluations were performed using the ECL western blotting detection reagent (GE Healthcare, Piscataway, NJ, USA) and ImageJ software (Bethesda, MD, USA).