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.