Cloning and purification of TAT-NF-Ya fusion proteins. (A) Design of human TAT-NF-Ya expression constructs. An N-terminal His-TAT-HA-NF-Ya expression construct was generated by cloning a human NF-Ya coding sequence into XhoI and EcoRI sites of the pTAT-HA vector. The C-terminally tagged TAT-HA-NF-Ya-His expression vector was produced by subcloning the TAT-HA-NF-Ya sequence into the pET-21b(+) vector. A GST-TAT-HA-NF-Ya was designed by introducing SalI-TAT-HA-NF-Ya-NotI PCR fragment into pGEX-6p1 plasmid. All PCR products were verified by sequencing. Fusion proteins were expressed using E coli strain Rosetta(DE3)pLysS (Novagen) and induced for 3 hours with 0.1mM isopropyl-β-D-thiogalactoside at 37°C. (B) Coomassie blue-stained SDS-PAGE showing affinity purification of His-TAT-NF-Ya. Western blots of His-TAT-HA-NF-Ya (C) and TAT-HA-NF-Ya-His (D) proteins probed with anti-HA antibody. (E-F) Purification of GST-TAT-HA-NF-Ya: Coomassie-stained gel (E) and immunoblot (F) with anti-GST antibody. Arrows indicate fusion proteins. NI indicates noninduced culture; CL, clear lysate; FT, flow through; W, wash; and E, eluate.