Ab129195

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.

The EZ-ChIP kit (Upstate, Lake Placid, NY, USA) was used to perform the ChIP assay based on a previously published protocol (Milne et al. 2009 (link)). In brief, BM-MSCs were resuspended in SDS lysis buffer and then sheared by sonication. The chromatin fragments were immunoprecipitated with antibodies against KDM1A (ab129195, Abcam), H3K9me3 (ab8898, Abcam) and the purified DNA was analyzed using RT-qPCR.

Total protein of histone was extracted using the histone extraction kit (#ab113476; Abcam). And, total protein of HL-1 cells was extracted using the cell lysis buffer RIPA and was quantified by BCA kits (Pierce, Rockford, IL, USA). The protein was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly (vinylidene fluoride) (PVDF) membrane (Invitrogen, Carlsbad, CA, USA). The membrane was blocked with 5% skim milk and incubated with the primary antibodies anti-KDM1A (1:10,000; ab129195; Abcam), anti-H3K4me3 (1:1,000; ab213224; Abcam), anti-H3 (1: 1,000; ab1791; Abcam), and anti-GAPDH (1: 2,500; ab9485; Abcam) and secondary antibody (1:2,000; ab6728; Abcam). H3 was used as the internal reference for H3K4me3, and GAPDH was used as the internal reference for other proteins. The protein bands were observed using an enhanced chemiluminescence kit (Pierce).

Primary antibody to LSD1 (ab129195, Abcam) was added and incubated overnight at 4 °C. PBS was rinsed three times for 5 min each time. Anti-Rabbit IgG-HRP (50–100 μL, Bio-Rad, USA) was added and incubated at 37 °C for 30 min, then washed with PBS three times for 5 min each. The samples were than exposed to two xylene solutions for 10 min each time. Each samples was sealed using neutral gum and examined by microscopy.

After washing twice with PBS, the undifferentiated and differentiated cells were harvested by the addition of ice-cold cell lysis buffer (Cell Signaling; #9803) and a protease inhibitor-phenylmethanesulfonyl fluoride (PMSF) (Cell Signaling; #8553) mixture into the culture system. Fifty micrograms of protein extracts were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene difluoride membranes. The membranes were blocked for 2 h at room temperature with 5 % nonfat dry milk in Tris-buffered saline and then stored in a box with the primary antibodies, which were diluted in 5 % nonfat milk/Tris-buffered saline, overnight with shaking at 4 °C. Subsequently, the membranes were washed thrice with TBS-T and then incubated with a horseradish peroxidase-conjugated secondary antibody with shaking for 1.5 h at 37 °C. The probed membranes were then incubated in 2 mL of ECL detection reagent for 4 min and subjected to image detection in a darkroom. GAPDH served as the control. The film was scanned according to the manufacturer’s suggestion, and a gel imaging system and an image analysis software were used to analyze the results. Anti-histone H3 peptide with dimethylated lysine 4 (H3K4me2; ab32356), anti-histone H3 (ab61251) and LSD1 (ab129195) antibodies were from Abcam.