Figure 1

K-RTA, but not EBV Rta, is recognized by the anti- O -GlcNAc antibody RL2. (A) Expression kinetics of K-RTA in Doxycycline (Dox)-treated 293TetKR cells (50 ng/ml) for the indicated times. C6: untreated 293TetKR at 6 h. (B) Protein stability of K-RTA in 293TetKR cells. The remaining amounts of K-RTA in 24 h Dox-treated 293TetKR cells were monitored between 4 and 24 h after Dox removal. ST: starting material. (C) The 20 potential O-GlcNAc sites present in the primary sequence of K-RTA composed of 691 amino acids were identified using the CBS YinOYang [45] and SVM [46] prediction programs. Ser and Thr residues scored positive in both programs are denoted by circles above the amino acid symbols. Open circles indicate that residues are only O-GlcNAcylated; solid circles represent residues that might also be phosphorylated. Thr-366 and Thr-367 are underlined. (D) Protein extracts from 293TetKR (K-RTA), 293TetNLSm (K-RTA defective in nuclear localization signal) and 293TetER (EBV Rta) cells were immunoprecipitated by an anti-O-GlcNAc antibody (RL2) followed by western blot analysis using the M2-FLAG antibody. Only K-RTA and NLSm were immunoprecipitated by anti-O-GlcNAc antibody.