Anti rps6

To confirm the interaction between HDA714 and ribosomal proteins in vivo, the proteins were extracted from Nicotiana benthamiana leaves expressing 35Spro:HDA714-GFP/ 35Spro:RPS3-FLAG, 35Spro:HDA714-GFP/35Spro:RPS6-FLAG or 35Spro:HDA714-GFP/35Spro:RPL7a-FLAG constructs using lysis buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-GFP agarose beads (ChromoTek) for 5 h at 4°C. After three washes with washing/dilution buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA), the co-immunoprecipitated proteins were separated by SDS-PAGE and detected with anti-GFP (Abmart, 1:1000 dilution) and anti-FLAG (Sigma, 1:1000 dilution) antibodies, respectively. For co-immunoprecipitation, ten-day wild type rice seedlings were ground into power by liquid nitrogen and extracted by lysis buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet™ P40 Substitute) and then incubated with anti-HDA714 (the full length HDA714 protein was used as antigen to produce polyclonal antibodies in rabbit) coated with protein-A magnetic beads (Thermo Fisher Scientific) for 5 h at 4°C. After four washes with PBST buffer, the co-immunoprecipitated proteins were separated by SDS-PAGE and detected with anti-RPS6 (Abcam, 1:1000 dilution) and anti-RPS3 (Abcam, 1:1000 dilution) antibodies.