Total Arabidopsis protein was extracted from 0.5 g of ground powder in 2 mL of extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % Triton X-100, 5 mM EDTA, 1x Protease inhibitor cocktail [Sigma-Aldrich] and 1 mM PMSF) and centrifuged at 4 °C at maximum speed for 10 min. 50 μL input was taken from the supernatant and 1 mL protein extract were incubated with 20 μL of anti-FLAG-M2-Agarose beads (Sigma-Aldrich A2220) for 1.5 h at 4 °C. And then washed three times with wash buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20). Samples were analyzed by SDS-PAGE and immunoblotting using HRP-conjugated anti-FLAG antibody (Sigma-Aldrich) and anti-H2A antibody (Abcam) as described above.
To detect the PIF4-RGA-H2A or IDD3-RGA-H2A complex, MYC-PIF4 or MYC-IDD3, HA-RGA and FLAG-H2A were transiently expressed in N. benthamiana leaves, and subsequent co-IP assays using rabbit anti-Myc polyclonal antibody conjugated agarose beads (Sigma-Aldrich A7470) were performed as described previously45 (link).
To detect the PIF4-RGA-H2A or IDD3-RGA-H2A complex, MYC-PIF4 or MYC-IDD3, HA-RGA and FLAG-H2A were transiently expressed in N. benthamiana leaves, and subsequent co-IP assays using rabbit anti-Myc polyclonal antibody conjugated agarose beads (Sigma-Aldrich A7470) were performed as described previously45 (link).
