H3k27me3

The ChIP assay was performed according to the manufacturer’s instructions for the EpiQuik Chromatin Immunoprecipitation (ChIP) Kit (Epigentek, Farmingdale, NY, USA). Primary antibodies against EZH2 (CST, USA), H3K27me3 (ABclonal, USA), H2AK119Ub1 (CST, USA), H3K4me3 (Affinity Biosciences, USA), H3K36me3 (Affinity Biosciences, USA), DNMT1 (Santa Cruz Biotechnology, USA) and DNMT3a (Santa Cruz Biotechnology, USA) were used for co-immunoprecipitation. RT-PCR was then conducted. Primers for the ChIP assay were determined according to a previous study^{18 (link), 23 (link)} and are listed in Supplemental Table S1. Internal normal mouse IgG and anti-RNA Polymerase II in the kit served as positive and negative control, respectively.

Cell or paraffin embedding section immunofluorescence was carried out as previously described. After fixation, permeabilization, antigen retrieval and blocking, and cell or section were incubated with corresponding primary antibodies at 4°C overnight. On the second day, the cell or section was washed with PBS three times, and Cy3- and Alexa Fluor 488-conjugated secondary antibodies were used for immunofluorescence or HRP-conjugated secondary antibody was used for immunohistochemistry. DAB was used to detect the primary antibody. The nuclear was stained with DAPI for immunofluorescence or hematoxylin for immunohistochemistry. The fluorescence images were taken with confocal microscopy (Nikon, Tokyo, Japan). The DAB-stained cardiac section was scanned by Pannoramic MIDI (3DHISTECH, Hungary). Fluorescence intensity was analyzed by ImageJ and normalized to control. Following antibodies were used at the intended dilution: H3K27me3 (Abclonal, Wuhan, China, A2363, 1:200), cTnI (ProteinTech, Wuhan, China, 66376-1-Ig, 1:200), 4-HNE (Bioss, Beijing, China, bs-6313R, 1:200), Cy3-conjugated goat-anti-rabbit secondary antibody (ServiceBio, Wuhan, China, GB21303, 1:200), and Alexa Fluro 488-conjugated goat-anti-mouse secondary antibody (ServiceBio, Wuhan, China, GB25301, 1:200).

The total proteins and nuclear proteins were extracted from the cells or tissue samples using a radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethylsulfonyl fluoride (PMSF). Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Vazyme, China). The proteins were then separated using SDS-PAGE and then electro-transferred to polyvinylidene difluoride (PVDF) membranes. After soaking in 10% skimmed milk powder for 1 h, the membranes were incubated overnight at 4 °C with specific antibodies: GFAP (ABclonal, Wuhan, China), GDNF (Abcam, Cambridge, UK), S100B (Abcam, Cambridge, UK), GAPDH (Antgene, Wuhan, China), H3K9me1 (A2358, ABclonal), H3K9me3 (A2360, ABclonal), H3K27me1 (A2361, ABclonal), H3K27me3 (A2363, ABclonal), and H3 (A2348, ABclonal). Thereafter, the membranes were incubated at room temperature with HRP-labeled secondary antibodies for 1 h. The intensities of the protein bands were determined using a chemiluminescence (ECL) kit (Vazyme).

N2a cells were lysed in RIPA buffer (Beyotime, P0013B) supplemented with 1× protease inhibitor cocktail (Roche). Total cell lysates were separated on 8–12% SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad). Membranes were blocked with TBST with 5% (w/v) non-fat dry milk for 4 h and probed with primary antibodies which were diluted with TBST and 5% (w/v) non-fat dry milk overnight in 4 °C. The primary antibodies were against RABV N protein (prepared by our lab, 1:5000), H3K27me3 (Abclonal Technology, Wuhan, China, A2363, 1:2000), H3K4me3 (Abclonal, A2357, 1:2000), H3K36me3 (Abclonal, A2366, 1:2000), H3 (Abclonal, A2348, 1:2000), EZH2 (CST, #5246, 1:2000), Flag tag (MBL, M185-3 L, 1:10,000), HA tag (MBL, M180-3, 1:10,000), PCP4L1 (ProteinTech, 25933-1-AP, 1:2000), or GAPDH (ProteinTech, 60004-1-Ig, 1:5000). After rinsing, membranes were probed with HRP-conjugated goat anti-mouse (Boster, Wuan, China, BA1051), goat anti-rabbit secondary antibodies (Boster, BA1055, 1:6000), or goat anti-mouse IgG light-chain secondary antibodies (Abbkine, A25012, 1:5000), then developed using BeyoECL Star kit (Beyotime, P0018A). Images were captured with an Amersham Imager 600 (GE Healthcare) imaging system.