Fig. 4.

Tat phosphorylation and the effect of CDK2 and PKR in cultured cells. a Mutation of Ser-16 or Ser-46 residue reduced HIV-1 Tat phosphorylation in cultured cells. Flag-tagged Tat, WT and S16A and S46A mutants were expressed in 293T cells and metabolically labeled with (³²P) orthophosphate. Tat protein was immunoprecipitated from cell lysates, resolved on 10% SDS-PAGE and exposed to Phosphor Imager screen. Tat expression was verified by Western blotting with anti-Flag antibodies. Lane 1, mock-transfected cells. Lane 2, WT Tat. Lane 3, Tat S16A mutant. Lane 4, Tat S46A mutant. The figure represents one of the three independent experiments. b Relative intensities of Tat and the mutants phosphorylation from three independent experiments. The mean ± SD are shown. *p < 0.01. c Label-free quantitative analysis of the high resolution MS spectra produced by Orbitrap MS scans for Tat by SIEVE 2.1 software. Average intensities of the indicated Tat peptide are shown with mean and standard deviations. d Quantification of non-phosphorylated and Ser-16 phosphorylated LEPWEHPGSQPK + Phospho(9) peptides derived from the data on c. Data are further adjusted to indicate the ratio of non-phosphorylated versus phosphorylated peptides. *p < 0.05