4.4. Transient Expression in Protoplast and Co-IP Assay

Rice protoplasts were prepared and transfected as previously described [37]. The protoplasts co-transformed with 20 μg plasmid (10 μg for each construct) were harvested by centrifugation at 5000 rpm for 30 s after incubation for 12 h. Approximate 2 × 106 protoplasts were homogenized in IP buffer [25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and Protease Inhibitor Cocktail (Roche, Palo Alto, NJ, USA)] and incubated for 30 min on ice with an intermittent vortex to ensure complete lysis. The mixture was centrifuged at 20,000× g for 10 min at 4 °C to discard debris. Thirty microliters of Protein A/G Agarose Beads (Abmart, Shanghai, China) were added to get the pre-clean extract by incubation for 3 h at 4 °C with gentle rotation. One microgram of antibodies were added to the pre-clean extract followed by incubation at 4 °C overnight. Thirty microliters of pre-equilibrated Protein A/G Agarose Beads were added and incubated for a further 3 h at 4 °C. The beads were collected by centrifugation at 100× g for 30 s at 4 °C and then washed five times with pre-chilled IP buffer. The proteins were eluted by boiling in SDS-PAGE loading buffer for 5 min before detection by western blot. The rat monoclonal anti-HA antibody was bought from Roche (11867423001) and the mouse monoclonal anti-FLAG antibody were from MBL International (M185-3L).

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