Anti h3k27me3

Sort purified lymphocytes from LNs and spleen (~5 x 10⁶ cells total) were fixed in 1% formaldehyde, resuspended in ChIP lysis buffer (1% v/v SDS, 10mM EDTA, 50 mM Tris-HCl) and sonicated to generate 200–1000 base pair fragments. Samples were then precleared using Protein A-agarose/salmon sperm DNA (Millipore, 16–157), split into 5 and incubated overnight with either 5 ug anti-H3K27me3, 3 ug anti-H3K4me3 or 4 ug anti-RNA polymerase II (all Invitrogen). A no antibody control and a total input positive control were also included. After washing, all samples (except the ‘total input’) were incubated with Protein A-agarose/salmon sperm DNA with rotation for 1 hour followed by a series of washes in low salt, high salt, lithium chloride, and TE buffers. DNA was eluted before crosslink reversal with 0.2M NaCl at 66°C overnight, followed by protein digestion with proteinase K (Promega). Immunoprecipitated DNA was extracted by phenol: chloroform:isoamyl (25:24:1) extraction and resuspended in HPLC water. For analysis, real-time PCR was used to measure the levels of ChIP-DNA, such that resulting cycle threshold (Ct) values were converted to copy number (#copies = 10⁵/2^Ct-17) and samples were normalised to their corresponding total inputs with background subtraction (no-antibody control).

ChIP was performed using ChIP Kit-One Step (Abcam) according to the manufacturer's protocol. 2 μL of anti-H3K27me2 (Invitrogen) or anti-H3K27me3 (Invitrogen) was used in the ChIP process. 2 μL of eluted DNA from ChIP was used in a 20 μL PCR reaction. The qPCR assay was performed as described above. The primers are shown in Table S2.

Proteins were extracted from cells (RIPA, P0013E, Beyotime Biotechnology) and mouse kidney (One Step Animal Tissue Active Protein Extraction Kit, C500006-0020, Sangon Biotech), and the protein lysates quantified through a bicinchoninic acid assay kit (Boster). Then, the proteins were boiled for 5 min at 100°C in loading buffer (20 μg) and fractionated in SDS-PAGE gel at 80 V for 30 min and then 100 V for 1 h. Next, the proteins were transferred to a nitrocellulose membrane (Boster) at 300A for 1.5 h. The membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% fat-free milk for 1 h. Then, the membranes were incubated with primary antibodies overnight at 4°C. After washing, the membranes were incubated with secondary antibodies for 2 h at room temperature. The membranes were incubated with a high-sig electrochemiluminescence kit (FDbio Science) and detected by using an image analysis system (Image-Pro Plus 6.0, Media Cybernetics).
All primary antibodies except anti-H3K27me2 (Invitrogen) and anti-H3K27me3 (Invitrogen) were from Abcam, and all secondary antibodies were from Sino Biological.