The RK and KKK in the WD of OsWRKY62 (W62WD) were substituted by A residues to generate W62WDAA (mutation RK), W62WDAAA (mutation KKK), and W62WD5A (mutation of both sites), by site-directed mutagenesis. The corresponding cDNAs of OsIMαΔIBB1a, OsIMαΔIBB1b, OsWRKY62, OsWRKY76, and their mutants were inserted respectively into a modified pGEX vector (pGEX-tag: 3 × myc or 3 × flag available at the C-terminus). The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and purified using Glutathione Sepharose 4B gel (GE Healthcare).
For pull-down assays, proper combinations of the recombinant proteins (1 µg each) were incubated with anti-Flag M2 affinity gel (Sigma) in IP buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 0.5% protease inhibitor cocktail (Sigma)) for 3 h at 4 °C. The beads were washed five times with the IP buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100) and then resuspended in 2 × SDS-PAGE loading buffer. The immunocomplexes were separated on 10% polyacrylamide gels and probed with anti-Flag and anti-Myc antibodies (Sigma).
For pull-down assays, proper combinations of the recombinant proteins (1 µg each) were incubated with anti-Flag M2 affinity gel (Sigma) in IP buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 0.5% protease inhibitor cocktail (Sigma)) for 3 h at 4 °C. The beads were washed five times with the IP buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 0.1% Triton X-100) and then resuspended in 2 × SDS-PAGE loading buffer. The immunocomplexes were separated on 10% polyacrylamide gels and probed with anti-Flag and anti-Myc antibodies (Sigma).