Ab134055

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.

Total proteins were isolated, and the concentration was determined using BCA method. After proteins was separated by using 10%–12% SDS-PAGE, proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA). The 1% BSA in TBS buffer was used to block the membranes and cultivated at 4°C overnight with primary antibodies against FOXP1 (1:5,000, ab134055, Abcam, Cambridge, UK), microtubule-associated protein 1A/1B-light chain 3 (LC3) (1:3,000, ab51520), P62 (1:1,000, ab207305), B-cell lymphoma 2 (Bcl-2) (1:1,000, ab59348), Bcl-2 associated X (Bax) (1:5,000, ab32503), AKT (1:500, ab8805), phospho (p)-AKT (1:1,000, ab38449), mTOR (1:10,000, ab134903), GAPDH (1:1,000, ab8245), β-actin (1:1,000, ab8226). Then incubating with the corresponding secondary antibodies. The protein signaling was visualized by ECL reagent. GAPDH and β-actin as loading controls and all antibodies were purchased from Abcam.