Ab183889

The total protein in tissues or cells was extracted by RIPA lysis (C0481, Sigma Aldrich, USA), lysed at 4 ℃ for 15 min, and centrifuged at 15,000 rpm for 15 min. The concentration was determined by bicinchoninic acid method (23227, Thermo, USA). The samples were subjected to gel electrophoresis, then moved into polyvinylidene fluoride membranes, which were blocked with 5% skim milk for 1 h, and incubated overnight with the primary antibodies (all from Abcam) against G9a (1:500, ab183889), FOXP1 (1:1,000, ab134055), GAPDH (1:5,000, ab8245). The next day, the membranes were rinsed with tris-buffered saline-tween containing 0.1% tween 20 and probed with secondary antibody IgG H&L-conjugated horseradish peroxidase (1:20,000, ab205718) for 1.5 h. The protein was developed by adding developer (NCI4106, pierce, Rockford, IL, USA). ImageJ 1.48u software (Bio-Rad, Hercules, CA, USA) was used for protein quantitative analysis, and the ratio of gray value of each protein to gray value of GAPDH was used for protein quantitative analysis. Each experiment was repeated three times.

Antibodies specific for RNase H1 (15606-1-AP, Proteintech, Wuhan, China), CHCHD2 (Co-IP:19424-1-AP, Proteintech, Wuhan, China), CHCHD2 (IF & WB:66302-1-Ig, Proteintech, Wuhan, China), G9a (Co-IP: ab183889, Abcam, Cambridge, UK), G9a (WB:66689-1-Ig, Proteintech, Wuhan, China), Sirt1 (07–131, Millipore, USA), α-tubulin (AF0001, Beyotime, Shanghai, China), H3 (AF0009, Beyotime, shanghai, China), H3 (ab1791, Abcam, Cambridge, UK), H3K9ac (ab10812, Abcam, Cambridge, UK), H3K9me2 (ab1220, Abcam, Cambridge, UK), DNA-RNA Hybrid S9.6 (ENH001, Kerafast, Boston, MA, USA), GST (66001-2-Ig, Proteintech, Wuhan, China), His (66005-1-Ig, Proteintech, Wuhan, China), and acetylated-lysine (3067, DIA-AN, Wuhan, China) were used in this study. Other antibodies used were as follows: Cy3 Goat Anti-Mouse IgG (H ​+ ​L) (A22210, Abbkine, Wuhan, China), FITC Goat Anti-Rabbit IgG (H ​+ ​L) (A22120, Abbkine, Wuhan, China), Cy3 Goat Anti-Rabbit IgG (H ​+ ​L) (AP132C, Sigma), and FITC Goat Anti-Mouse IgG (H ​+ ​L) (F0257, Sigma).