Anti ha sc 7392 hrp

Pistils in various genetic backgrounds were collected, and the cell fractionation was carried out as described above. Nuclear protein extracts were 4-fold diluted by extraction buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5% Triton X-100, 1 mM PMSF, proteinase inhibitor cocktail, and 25 μM MG132) and incubated with anti-HA agarose beads (Sigma) or anti-FLAG magnetic beads (Sigma) at 4°C for 2 h and then washed four to six times by extraction buffer. Immunoprecipitated proteins and nuclear protein extracts as inputs were resolved by SDS–polyacrylamide gel electrophoresis and detected by the corresponding antibody (anti-FLAG: F3165, sigma, 1:5,000 dilution; anti-HA: sc-7392 HRP, Santa Cruz, 1:2,000 dilution; anti-H3: ab1791, abcam, 1:3,000 dilution). For IP-MS, immunoprecipitated proteins were eluted and analyzed by TripleTOF 5600 System (AB Sciex).

Siliques 4 days after pollination in various genetic backgrounds were collected, and total proteins were extracted by extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5% Triton X-100, 1 mM PMSF, proteinase inhibitor cocktail, and 25 μM MG132). Protein extracts were incubated with anti-HA agarose beads (A2095, Sigma) at 4 °C for 2 h, and then washed four times by extraction buffer. Immunoprecipitated proteins and total protein extracts as inputs were resolved by SDS-polyacrylamide gel electrophoresis and detected by anti-GFP antibody (anti-GFP: ab290, Abcam, 1:2000 dilution) and anti-HA HRP (anti-HA: sc-7392 HRP, Santa Cruz, 1:2000 dilution).