Anti histone h3

The NE‐PER nuclear and cytoplasmic extraction reagents (78833; Thermo Scientific) were used to separate and isolate cytoplasmic and nuclear proteins. In order to obtain the correct protein concentration, extraction was performed from 8.5 × 10⁶ HEK293T cells per sample. Isolation was performed according to the manufacturer's instructions. Anti‐Histone H3 (against the nuclear loading control protein, 17168‐1‐AP; Proteintech) and anti‐GAPDH (against the cytoplasmic loading control protein, Santa Cruz, 6C5) antibodies were used to check the efficiency of the separation of two fractions.

1 μg of isolated proteins underwent separation on a 15% or 10% SDS-PAGE prior to transfer onto PVDF membranes (BIORAD, Shanghai, China 162-0177), which then underwent a 2h blocking in 5% skimmed milk, followed by an ON incubation at 4 °C with anti-Histone H3 (Proteintech, 17168-1-AP, Planegg-Martinsried, Germany) (1:10,000), anti-Phospho-Histone H3 (Ser10) (Cell Signaling Technology, 13576S, Damvers, MA, USA) (1:1000), Anti-Di-Methyl-Histone H3 (Lys9) (Abcam, ab1220, Cambridge, UK) (1:2000), and anti-acetyl-Histone H3 (Lys18) (PTM BIO, PTM-158) (1:2000). This was followed by four PBST-rinses, and subsequent 2 h secondary antibodies incubation (Goat Anti-Rabbit IgG H&L (HRP, Abcam, ab6721, 1:10,000, Cambridge, UK) and Goat Anti-Mouse IgG H&L (HRP, Abcam, ab6789, 1:10,000), Cambridge, UK) at RT. Again, the membranes were rinsed four times in PBST buffer before employing the High-sig ECL Western Blotting Substrate (Tanon) to identify protein bands.

Cells were lysed with Western and IP lysis buffer (P0013J, Beyotime) with a protease inhibitor cocktail (Roche). Total cell lysates were incubated with 30 μl of Protein Agarose (Beyotime, P2012) and IgG (Beyotime, 1:50) for preclearing 2 h at 4 °C, and immunoprecipitation was then performed with an indicated antibody for 4 h at 4 °C. As a negative control, Rabbit IgG (Beyotime, A7016, 1:50) or Moues IgG (Beyotime, A7028, 1:50) was used. Separation of proteins by SDS-polyacrylamide gel electrophoresis (PAGE) and transfer to nitrocellulose membranes (Millipore) were carried out. The antibodies were used as follows: anti-RNF31 (ab125189, Abcam, 1:2000); anti-HA (MMS-101R, COVANCE, 1:2000); anti-Flag (20543–1-AP, Proteintech, 1:2000); anti-YAP (14,074, Cell Signaling Technology, 1:2000; SC-101199, Santa Cruz, 1;500); PD-L1 (13,684, Cell Signaling Technology; 66,248–1-Ig, Proteintech); anti-Tubulin (11224–1-AP, Proteintech, 1:5000); anti-Histone-H3 (17168–1-AP, Proteintech, 1:1000) anti-Myc (Ab9106, Abcam, 1:2000); and anti-β-actin (8H10D10, Cell Signaling Technology, 1:10000). AffiniPure goat anti-mouse peroxidase-conjugated antibody IgG-HRP (A0216, Beyotime, 1:5000) or goat anti-Rabbit IgG-HRP (A0208, Beyotime, 1；5000) secondary antibodies was incubated with the membranes after three washes with PBST. The signals were detected with the ECL Kit (Meilunbio, #MA0186).

In brief, acutely isolated soleus muscle was labeled with R-BTX in cold PBS for 5 min, and separated into SR and NSR according to the localization of R-BTX signal under a fluorescence microscope. Tendons at the muscle terminus were excluded. Samples were lysed in the lysis buffer (50 mM Tris–HCl, pH 7.8; 150 mM NaCl; 1% Triton X-100; 0.1% SDS; 1 mM EDTA; 5 mM NaF; 2 mM Na₃VO₄; 1 mM PMSF; and protease inhibitor cocktails) and resolved by SDS-PAGE for immunoblot. Primary antibodies: anti-Kdm1a (1:1000, CST, 2184), anti-H3K4me2 (1:1000, Abcam, ab8580), anti-Histone H3 (1:1000, Proteintech, 17,168–1-AP), anti-Neurofilament-L (1:1000, CST, 2837), anti-Myl4 (1:1000, Proteintech, 67,533–1-Ig), anti-α-tubulin (1:5000, Proteintech, 66,031–1-Ig), anti-GAPDH (1:5000, Proteintech, 60,004–1-Ig). Secondary antibodies: HRP-conjugated goat anti-mouse/rabbit IgG (1:5000, Thermo Fisher, 31,430 and 31,460). Immunoreactive bands were visualized by using SuperSignal West Femto maximum sensitivity substrate (34,095, Thermo Fisher). Autoradiographic films were scanned with a Canon MF3010 scanner.

Proteins were extracted from liver tissues using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China), in accordance with the manufacturer's instructions. Total protein content was determined using the BCA Protein Assay Kit (Beyotime). Equal amounts of protein were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) on 5% to 10% polyacrylamide gels and then transferred onto PVDF membranes (Millipore, Bedford, MA). The membranes were blocked with 10% nonfat milk in TBST (Tris‐buffered saline, 0.1% Tween 20) and incubated with anti‐NF‐κB p65 and anti‐IκB (Cell Signalling Technology, Danvers, MA.), anti‐histone H3, and anti‐β‐actin (1:1,000 dilution; Proteintech, Rosemont, IL) antibodies at 4°C overnight. Subsequently, membranes were washed three times with TBST for 10 min, followed by incubation with the corresponding secondary antibodies (1:3,000 dilution; Zhongshan Goldenbridge, Beijing, China) for 2 hr at 37°C. Finally, protein bands were visualized using an enhanced chemiluminescence detection kit (Beyotime). Histone H3 and β‐actin served as internal controls for nucleoprotein and cytoplasmic proteins, respectively.

Tissues or cultured cells were lysed with RIPA lysis buffer with protease inhibitors and phosphatase inhibitors (Beyotime, Shanghai, China) using standard methods. After centrifugation and quantification, 25 µg of protein was loaded for blotting with the indicated specific antibodies. A NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, Waltham, USA) was used to isolate nuclear and cytoplasmic fractions according to the manufacturer’s protocol. The following antibodies were used: anti-Sox9 (1:1000, Abcam, Cambridge, UK), anti-HNF4α (1:2000, Abcam, Cambridge, UK), anti-TERT (1:1000, Novus, Colorado, USA), anti-E-Cadherin (1:1000, Abcam, Cambridge, UK), anti-Vimentin (1:1000, CST, Danvers, USA), anti-CK19 (1:1000, Abcam, Cambridge, UK), anti-LGR5 (1:1000, Abcam, Cambridge, UK), anti-Epcam (1:1000, Abcam, Cambridge, UK), anti-P65 (1:1000, CST, Danvers, USA), anti-p-P65 (1:1000, CST, Danvers, USA), anti-Bcl3 (1:100, Abcam, Cambridge, UK), anti-Histone H3 (1:1000, Proteintech, Rosemont, USA), anti-YAP1 (1:1000, CST, Danvers, USA), anti-CTGF (1:1000, Abcam, Cambridge, UK), anti-Cyr61 (1:1000, Abcam, Cambridge, UK), anti-His (1:5000, Proteintech, Rosemont, USA), anti-Flag (1:2000, Proteintech, Rosemont, USA), anti-Myc (1:1000, Proteintech, Rosemont, USA), anti-Ubiquitin (1:1000, CST, Danvers, USA), and anti-β-actin (1:4000, Bioworld, MN, USA).