Ni columns

Twenty SR proteins from O. sativa were selected for the screening of specific RNA-binding proteins. Individual primers for these 20 SR proteins were designed according to their cDNA sequences. The primers were listed in Supplementary Table S2. The cDNA was amplified by PCR and cloned into the PET30a vector for protein expression. Protein expression was induced by IPTG (0.6 mM) for six hours at either 28 °C or 37 °C. To maintain the activity of the proteins, Escherichia coli cells expressing the SR proteins were lysed by lysozymes and purified on Ni columns (Qiagen, Germany). The purified SR proteins were then used for EMSA.

Glutathione S-transferase (GST) fusion proteins were expressed in BL21 (DE3)pLysS E. coli and purified with Glutathione Sepharose 4B following the manufacturer’s protocol (GE). His-tagged proteins were expressed in BL21 (DE3)pLysS E. coli using pET28A vectors and purified using Ni-columns according to the manufacturer’s protocol (Qiagen). Purified proteins were dialyzed against 1 × Dulbecco’s PBS (DPBS) buffer. Unless specified, pull-down assays and western blotting were performed according to standard protocols. Cellular lysates, prepared using 1 × DPBS containing 1% Triton X-100, 0.5–1 mg/ml total cellular protein (dependent on expression), in a total volume of 1 ml, were mixed with 20 μg GST fusion protein bound to beads and incubated at 4°C while rotating for 2 h. The beads were spun down and washed thrice using lysate buffer. Bound proteins were eluted using 1 × loading buffer and detected by western blotting.