H4r3me2a

Whole cultured cells were homogenized in 0.1% SDS and 1 mM PMSF (phenylmethylsulfonyl fluoride) and centrifuged at 12,000 g for 15 min. The total histone proteins were extracted from ESCC cells using a Histone Extraction Kit (Abcam, ab113476), following manufacturer’s protocol. Protein extracts were subjected to SDS-PAGE and analysed using the following primary antibodies: PRMT1 (Abcam, ab73246), H4R3me2a (Active Motif,), H4R3me2s (Active Motif), histone H3 (CST) and GADPH (Abcam, ab8245). Then the membranes were incubated with secondary antibodies (CST,7076,7074) at room temperature for 1 h. The dilution ratio was determined according to the recommended instructions. Protein levels were detected by the Image-Pro Plus 6.0 system (Bio-Rad,1708265). Quantification of bands intensity was measured using ImageJ software (version 1.34). All experiments were performed in triplicates.

ChIP assays were performed as described previously^{8 (link)} using antibodies specific for PRMT1 or H4R3me2a (Active Motif). The primers used for ChIP-qPCR are ATF5 promoter (Forward: TGGGAAGGAAAGGCTCGGAT; Reverse: CGGCGACACTGTAGTGAGAA) and CITED2 promoter (Forward: CTCAGAAGAGCCCAGTGTAGCA; Reverse: GGATGAGGTATGTTGGAAAGCAGA).

For protein isolation, the intestinal crypts in the lysis buffer above were homogenized and incubated for 10 min at 4°C, centrifuged (14 000 rpm, 4°C, 15 min) and the supernatant was collected. For Western blot analysis, 10 μg proteins were electrophoresed on a 10%-20% Tris-Glycine gel (Thermo Scientific) and transferred to polyvinylidene fluoride (PVDF) membranes (Immuno-Blot PVDF, BioRad) with TransBlot (BioRad) for 10 min. The membranes were blocked with 4% BSA in TBS-0.1%Tween 20 and then incubated with primary antibodies, rabbit anti-PRMT1 (Upstate, 1:1000 dilution), H4R3me2a (Active Motif, 1:1000 dilution), or mouse β-actin (R&D, 1:2000 dilution) as a loading control at 4°C overnight, followed by incubation with Goat anti-Rabbit IRDye 680® or Goat anti-mouse IRDye® 800 secondary antibody (Odyssey) for 1 hour at room temperature. The proteins of interest were detected by using Odyssey® infrared imaging system (Li-Cor Bioscience), and protein bands were quantified by using Li-Cor software.