Figure 1.

Analysis of HIV-1 Tat phosphorylated by CDK2/cyclin E in vitro. A, Tat is phosphorylated by CDK2. Recombinant Tat was phosphorylated in vitro by purified CDK2/cyclin E (lane 1), by HeLa nuclear extract (lane 2) or by CDK2-depleted HeLa nuclear extract (lane 3). Tat was resolved by 12% SDS Tris-Tricin PAGE. The gel was stained with Coomassie blue (upper panel) and exposed to Phospho Imager screen (lower panel). B, HPLC profiles of Tat peptides after trypsin cleavage. Recombinant Tat was phosphorylated in vitro by purified CDK2/cyclin E, resolved by 12% SDS Tris-Tricin PAGE, and subjected to in-gel trypsin digestion. The eluted peptides were resolved by reverse phase chromatography on μRPC C2/C18 ST 4.6/100 column. No Tat, mock trypsin digest without Tat. Tat, digest of non-phosphorylated Tat. (Phospho)-Tat, digestion of phosphorylated Tat. I and II, peaks identified in the elution profile of phosphorylated Tat that were subjected to MALDI TOF/TOF mass spectrometry.