Anti ezh1

For histone-specific Western blotting, histones were enriched by lysing cells in an acid lysis buffer (10 nM HEPES, 1.5 mM MgCl₂, 10 mM KCl). For all other Western blots protein lysates were generated using RIPA Buffer (Cell Signaling, #9806). Protein lysates and histone extractions were separated by SDS-PAGE, transferred to a PVDF membrane, and detected by Western blotting (WB). Membranes were blocked with 5% BSA in 1X TBS with 0.01% Tween20 (TBST) for 1 h at room temperature and incubated with primary antibodies in 5% BSA in TBST overnight at 4 C (1:1,000 anti-EZH2 (Cell Signaling, D2C9), 1:1,000 anti-EZH1 (Proteintech, #20852-1AP); 1:1,000 anti-EZH1 (Reinberg lab^{21 (link)}), 1:1,000 anti-H3K27me3 (EMD Millipore, 07-449), 1:5,000; anti-β-actin (GenScript, A00702), 1:10,000 anti-H4 (Abcam, ab10158)). The next day the membrane was washed three times with TBST, incubated for 1 h with the corresponding secondary anti-Rabbit HRP (Invitrogen, #31458) and anti-Mouse HRP (Invitrogen, #SA1-100), and washed three times with TBST. The membranes were developed using Pierce™ ECL Western Blotting Substrate kit (Thermo Scientific, #32106) and exposed to autoradiography films following development in AFP Mini-Med 90 X-Ray Fil Processor. ImageJ software was used for densitometry quantification of WB bands.

The amount of 40-50 μg protein prepared from mouse myocardium or NMVCs was usedin a standard western blot analysis. The polyvinylidene fluoride (PVDF) membrane binding sample protein was incubated with a high affinity anti-Col1a1 antibody (1:1000), anti-Col3a1 antibody (1:1000), anti-α-SMA (1:2000) (Santa Cruz Biotechnology, USA), anti-EZH1 (1:1000), anti-EZH2 (1:1000), anti-PPAR-γ (1:1000) (Proteintech, Rosemont, IL, USA), anti-p-NF-κB P65 (1:1000), anti-NF-κB P65 (1:1000)(Cell SignalingTechnology, USA), respectively. An anti-GAPDH antibody (1:2000) (Santa CruzBiotechnology, USA) was used to detect the level of GAPDH as an internal control. Protein was visualized using the ECL Plus detection system (GE Healthcare, Waukesha, WI).