Amersham hybond

Total histones were extracted from the frozen panicle samples using the EpiQuik Total Histone Extraction kit (Epigentek, Cat.# OP-0006) following the protocol. Concentration of the eluted protein was measured by the Bradford method according to the manufacturer’s instructions (Bio-Rad protein assay, Hercules, CA, USA). For immunoblotting, the histone samples were diluted with SDS loading buffer, and 20 μg proteins of each sample were separated by 16.5% SDS-PAGE and electro-blotted onto PVDF blotting membrane (Amersham Hybond, Cytiva). The blot was probed with anti-acetyl-histone H3 antibody (Millipore, Cat.# 06-599, 1:1000 dilution), anti-acetyl-histone H3K37 antibody (Active Motif, Cat.# 61 587, 1:1000 dilution), or anti-histone H3 antibody (Sigma, Cat.# SAB5701101, 1:1000 dilution).

Western blotting was performed by standard procedures using Novex Tris-Glycine buffered gradient WedgeWell gels (8–16%) (Invitrogen – Thermo Fisher Scientific) in a Mini Cell apparatus. All protein transfers were performed overnight onto Amersham^™ Hybond^™ (Cytiva) PVDF membranes 0.45 µm pore size. Chemiluminescence from HRP-conjugated secondary antibodies (1:20.000 dilution) (Thermo Fisher Scientific) was developed using SuperSignal^™, West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Antibodies used: Mouse anti-Streptag II antibody (Antibodies Online) ABIN7250959, (1:1.000) Mouse anti-STAT3 (Cell signaling technologies, CST-9139) (1:1000), Mouse anti-HuR (Santa Cruz Biotechnology) (SC-365338) (1:5000), Rabbit anti-p53 (Cell signaling technologies, CST-2527) (1:500), and Rabbit anti-HIPK3 (Abcam Ab72538) (1:500). Acquisition and quantifications of band intensities were performed using a Licor^™ OdysseyFc^™ scanner and Image Studio version 5.2 software. Band intensities were normalized to abundant house-keeping protein HuR or in some cases total protein assessed by Revert Stain (Licor^™). The normalized band intensities are based on 3 biological replicates (quantified western blots - plotted data). Error bars represents standard deviations from means (n=3).