A histone H3K4me1-specific binding protein is required for siRNA accumulation and DNA methylation at a subset of loci targeted by RNA-directed DNA methylation

ABSTRACT: The RDM15 genomic sequence was fused in frame to the 3xFLAG tag and 3xMYC tag, and the fused sequences were inserted into the pCambia1305 backbone and were transformed into Arabidopsis plants, respectively. A 5-g quantity of flower tissue was harvested from transgenic plants and ground in liquid nitrogen. The proteins were extracted in 25 mL of lysis buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.1% Nonidet P-40, 0.5 mM DTT, 1 mM PMSF, and 250 ul of plant cell protease inhibitor (Sigma)]. After the proteins were extracted, anti-Flag M2 (Sigma; F3165) or anti- Myc (Sigma; M5546) was added and incubated at 4 °C for 2–3 h. The resins were then washed with the lysis buffer at least five times. The protein samples were sent to the Core Facility for Proteomics at the Shanghai Center for Plant Stress Biology (PSC) for affinity purification and mass spectrometry.

ORGANISM(S): Arabidopsis Thaliana

SUBMITTER: Zhaobo Lang  

PROVIDER: PXD025147 | iProX | Thu Apr 01 00:00:00 BST 2021

REPOSITORIES: iProX