Anti crotonyllysine

Histone-enriched fractions were extracted from seedlings of Arabidopsis, rice, maize, and tobacco plants using EpiQuik Total Histone Extraction Kit (Epigentek USA, OP-0006-100). The histone-enriched fractions were used for immunoblot analysis; antibodies used in this study were anti-H3 (ab1791; Abcam), anti-H3K9ace (07-352; Millipore), anti-butyryllysine (PTM-301; PTM Biolabs), and anti-crotonyllysine (PTM-501; PTM Biolabs). Immunoblotting results were quantified using ImageJ (v1.6.0_24).

Two grams of rice seedling leaves was cross-linked by 1% formaldehyde and used for chromatin extraction. After sonication, chromatin fragments were incubated with antibody (anti-H3K9ac, anti-Kbu, and anti-Kcr)-coated beads (Invitrogen/Life Technologies; 10001D) overnight. Specificity of anti-Kbu and anti-Kcr was tested by dot blots (Additional file 1: Figure S8). After extensive washing, immunoprecipitated chromatin was de-cross-linked and retrieved for qPCR or sequencing. anti-H3K9ace (07-352; Millipore), anti-butyryllysine (PTM-301; PTM Biolabs), and anti-crotonyllysine (PTM-501; PTM Biolabs) antibodies were used.

Thirty micrograms of total protein was loaded into each well of a 12% SDS–PAGE gel, and the proteins were separated by electrophoresis. The proteins were then transferred to PVDF membranes (Millipore; Bedford, MA, United States). After blocking, the membranes were incubated with anti-crotonyl-histone H2B (Lys20) (Cat#: PTM-512, PTM Bio, Hangzhou, China) (1: 2000), anti-crotonyl-histone H2B (Lys34) (Cat#: PTM-514, PTM Bio, Hangzhou, China) (1: 1000), anti-crotonyllysine (Cat#: PTM-502, PTM Bio, Hangzhou, China) (1: 200), anti-2-hydroxyisobutyryl-histone H3 (Lys14) (Cat#: PTM-881, PTM Bio, Hangzhou, China) (1: 1000), anti-2-hydroxyisobutyryl-histone H3 (Lys79) (Cat#: PTM-845, PTM Bio, Hangzhou, China) (1: 1000), anti-2-hydroxyisobutyryl-histone H4 (Lys5/8/12) (Cat#: PTM-850, PTM Bio, Hangzhou, China) (1: 1000), anti-GAPDH (Cat#: PTM-6620, PTM Bio, Hangzhou, China) (1: 2000) and anti-VCP (Cat#: PTM-5823, PTM Bio, Hangzhou, China) (1: 2000) overnight at 4°C. Then, the membranes were incubated with HRP-labeled secondary antibodies. Finally, the protein bands were visualized using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Silver Spring, MD, United States) and measured with Quantity One software (Bio-Rad Laboratories, Hercules, CA, United States).