Ab176877

A Magna ChIP™A/G One‐Day ChIP kit (cat. No. 17–10085; EMD Millipore Corp., Billerica, MA, USA) was used according to the manufacturer's instructions. In short, fresh frozen cortex tissues were cut into 1–3 mm³ fragments. The tissue fragments were loaded into 50‐ml tubes, added with 10 ml PBS and then formaldehyde till a final concentration of 1%, rotated at room temperature for 10 min of crosslinking, and then neutralized with glycine for 5 min. The tissues were destructed by SDS lysis buffer (1% SDS, 10 mM EDTA and 50 mM Tris‐HCl; pH 8.0) and treated using a high‐intensity ultrasonic processor on ice at 150 Hz. After centrifugation and collection of the supernatant, an equal amount of chromatin was used for immunoprecipitation at 4℃ overnight. The specific antibodies against LSD1 (ab129195; Abcam) or H3K4me1 (ab176877; Abcam) were used. Anti‐rabbit IgG was used as control, and total chromatin was used as input. After incubation with magnetic beads, the immunoprecipitates were collected. The magnetic beads were washed, and the chromatin was eluted by the proteinase K mixture in ChIP elusion buffer. The TGIF2 expression in the DNA fragments immunoprecipitated by LSD1 or H3K4me1 was examined by qPCR.

Cellular total protein was extracted with radioimmunoprecipitation assay buffer containing protease inhibitors. For histone extraction, cells were collected by refrigerated centrifugation at 300 × g for 10 min. The pellet was incubated for 30 min in hypotonic lysis buffer [10 mM Tris-Cl (pH 8.0), 1 mM KCl, 1.5 mM MgCl₂, 1 mM DTT and protease inhibitors]. The nuclei were resuspended in acid-extraction buffer (0.4 N H₂SO₄), and the extracted histones were precipitated with trichloroacetic acid and resuspended in deionized water. Equal amounts of protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated with primary antibodies, including anti-TBX1 (ab109313, Abcam), anti-H3K4me1 (ab176877, Abcam), anti-H3K4me2 (ab32356, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-histone H3 (ab176842, Abcam) and anti-β-actin (BS6007MH, Bioworld Technology), overnight at 4°C and then with horseradish peroxidase-conjugated secondary antibodies (Sinopept) for 1 h at room temperature. Visualization was performed using an enhanced chemiluminescence detection system (Millipore). Experiments were repeated in triplicate.

Cells and exosomes were lysed by RIPA lysis buffer, and the total protein was extracted. The denatured protein sample (20 μg) was separated by 12% SDS-PAGE and transferred to the polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk and probed with primary antibodies (Abcam) against CD133 (1:1000, ab19898), Nestin (Mouse, 1:2000, ab254048), GFAP (1:10,000, ab7260), Oct4 (1:1000, ab181557), Sox2 (1:1000, ab92494), MLL4 (Mouse, 1:1000, ab56770), H3K4me1 (1:5000, ab176877), H3K9me3 (1:1000, ab8898) and β-actin (1:5000, ab179467). The peroxidase-labeled goat anti-rabbit or anti-mouse IgG (H+L) served as secondary antibody. The blots were developed by chemiluminescence system (Millipore, Billerica, MA). The density of western blot band was quantified by ImageJ software, and normalized to β-actin.

ChIP kit (Millipore) was adopted to test the binding of PRDM1 enhancer to IL-33 promoter. Upon reaching about 70–80% confluence, cells were cross-linked with 1% formaldehyde for 10 min and then randomly broken into fragments by ultrasonication. The supernatant was collected after centrifugation at 13,000 rpm and 4 °C. The supernatant was severally incubated with positive control antibody RNA polymerase II, anti-human IgG and rabbit anti-H3K4me1 (1:100, ab176877, Abcam) and H3K9me3 (1:100, ab8898, Abcam) at 4 °C overnight. Endogenous DNA-protein complex was precipitated by Protein Agarose/Sepharose and centrifuged to remove the supernatant, and nonspecific complex was rinsed. The cross-linking was reversed at 65 °C overnight. DNA fragments were purified and recovered by phenol/chloroform extraction. The expression of PRDM1 enhancer and IL-33 promoter was determined by RT-qPCR.