Figure 2.

Interaction of OsRAA1 with OsRPT4. A, OsRAA1 interacts with OsRPT4. GST-OsRAA1 fusion protein and GST were expressed and purified from Escherichia coli, adsorbed onto GST-Sepharose 4B, and incubated with His-OsRPT4. After being washed, the protein-bound protein was fractionated by 12% SDS-PAGE and underwent western-blot analysis with antiserum of RAA1 (lane 4), GST antibodies (lane 2), or RPT4 antibodies (lanes 3 and 5). One-tenth of the total input of the E. coli total protein inducing His-RPT4 expression was loaded onto SDS-PAGE gels and detected by RPT4 antibodies (lane 1). B, Western-blot results of in vivo coimmunoprecipitation confirming the interaction between OsRAA1 and OsRPT4. Protein extracts from transgenic rice roots (left and middle) or transgenic yeast cells (right) were immunoprecipitated with 3 μL of anti-OsRAA1 (lanes 1, 3, and 5) or control preimmune serum (lanes 2, 4, and 6). The immunoprecipitated solution then underwent 12% T and 16.5% T, 3% C SDS-PAGE, then was incubated with anti-RPT4 (1:400; lanes 1, 2, 5, and 6) and anti-OsRAA1 (1:400; lanes 3 and 4; top images). The bottom images show Coomassie Brilliant Blue staining of loading controls. Molecular mass markers are indicated at left. Stars indicate immunoglobulin heavy chains. C, Localization of OsRAA1-GFP fusion protein in onion cells. D, BiFC view of OsRAA1 and OsRPT4 interaction in Chinese cabbage epidermal cells on transient expression assay. Corresponding differential interference contrast images are shown at top. Bars = 20 μm.