Figure 2.

OsSRT1 interacts with OsGAPDH1 and OsGAPDH2. (A) Pull-down assays of interaction between OsSRT1 and OsGAPDH1 (left) and OsGAPDH2 (right). OsSRT1–6XHis fusion protein or 6XHis alone (from PET32a vector) was incubated with OsGAPDH1-GST or OsGAPDH2-GST followed by immunoprecipitation with anti-His antibody and analyzed by immunoblots with anti-GST and anti-HIS. (B) Co-IP assays of in vivo interaction between OsSRT1 and OsGAPDH1 (upper part) and OsGAPDH2 (lower part) in tobacco cells. Nuclear proteins were extracted from tobacco leaves that were injected with Agrobacteria transformed with 35S:OsSRT1-FLAG together with binary vectors 35S:GAPDH1-GFP or 35S:GAPDH2-GFP, and immuno-precipitated with anti-FLAG or with IgG as controls, followed by immunoblots with anti-GFP. (C) Co-IP assays of in vivo interaction between OsSRT1 and OsGAPDH1 (upper part) and OsGAPDH2 (lower part) in rice cells. Nuclear proteins were extracted from wild type or 35S:OsGAPDH1-GFP transgenic rice seedlings (10-day-old), or from rice seedling protoplasts transiently transfected with 35S:OsGAPDH2-GFP or with 35S:GFP, and immuno-precipitated with anti-GFP, followed by immunoblots with anti-OsSRT1 and anti-GFP. (D) Interactions between OsSRT1 and OsGAPDH1/2 occur in the nucleus. Rice protoplasts were transfected by OsGAPDH1-pVYCE (left) or OsGAPDH2-VYCE (right) with OsSRT1-pVYNE or pVYNE alone. Reconstructed fluorescent YFP by the interactions was visualized by excitation at 488 nm. GHD7-CFP, a rice nuclear localized protein, was used as a control. Chlorophyll auto-fluorescence was excited at 543 nm. Bars = 10 μm.