Ab17726

For protein isolation after treatments, cells were lysed in the RIPA buffer (Beyotime, China). And then, we quantified the whole proteins and boiled in sodium dodecyl sulfate. The SDS-PAGE gel and nitrocellulose membranes (GE Healthcare) were utilized to separate the proteins and be transferred onto by separated proteins, respectively. We used primary antibodies to incubate the membranes overnight at 4°C. After washing the membranes for five times by phosphate-buffered saline supplemented with Tween 20 (PBST), the corresponding horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) were used to incubate the membranes for 1 h at room temperature. The Super Signal West Femto kit (Pierce, Rockford, IL) was utilized to bring the bands on the membranes into visualization in the final. The primary antibodies and secondary antibody were used as following: rabbit anti-FOXP4 (1:1000, Abcam, ab17726), rabbit anti-GAPDH (1:5000, Abcam, ab181602) and IRdye 800-conjugated goat anti-rabbit IgG. We used GAPDH as the endogenous control in this assay.

Cells were cross-linked with 1% formaldehyde for 15 min and were lysed with 500 µl lysis buffer (20mM Tris HCl pH 8, 1% SDS, 50mM NaCl) containing 1x protease inhibitor cocktail (with EDTA). Chromatin samples were sheared to a length of 300-500 bp via sonication. Following centrifugation, supernatants were collected and 2 µg of antibody (#ab16645 [FOXP1], #ab17726 [FOXP4], Abcam) was added to chromatin to immunoprecipitate overnight. Dynabeads Protein G (10003D, Thermofischer) was added to antibody chromatin complexes for 2h. Next, protein G bead-chromatin complexes were washed twice with dilution buffer (20mM Tris HCl pH 7.6, 1% Triton X-100, 150mM NaCl) and once with washing buffer (PBS, 0.02% Tween-20, pH 7.4), as specified by manufacturer’s instructions. Proteinase K (P8107S, New England Biolabs) was added to immunoprecipiated and input chromatin; samples were then incubated at 45°C for 2h. Subsequently, chromatin was heated at 64°C for 4h for reversal of formaldehyde crosslinking. DNA fragments were purified using a PCR purification kit (FAGCK001-1, Favorgen) and were analyzed by qPCR. Antibodies used for ChIP are the same as for western blotting. Primer pairs used for ChIP assays are listed in Table S4.