Figure 3
Contribution of PP2A and PP1 to Tat-activated transcription in vitro. A, In vitro transcription reactions were carried with the indicated amounts of recombinant Tat. Lane 1, no DNA template; lane 2, no Tat added; lanes 3–5, Tat added at 10 ng, 50 ng and 100 ng correspondingly. Transcription product was resolved on 5 % Urea-PAGE, exposed to the PhosphoImager screen and quantified. B, Inhibition of PP1 and PP2A by okadaic acid in phosphorylase-a dephosphorylation assay. PP1 and PP2A were inhibited by okadaic acid with IC50 = 70 nM and 0.4 nM concentration of inhibitor respectively. C, Okadaic acid inhibits basal and Tat-activated transcription. Lane 1, no DNA template; lane 2, no Tat added; lane 3, transcription with 50 ng of Tat; lanes 4 and 5, transcription in the absence of Tat and with 10 nM or 1 μM of okadaic acid; and lanes 6 and 7, transcription in the presence of 50 ng of Tat and with 10 nM or 1 μM of okadaic acid. Transcription products were resolved on 5 % Urea-PAGE, exposed to the PhosphoImager screen and quantified. D, NIPP1 inhibits Tat-activated transcription. Lane 1, no DNA template; lane 2, no Tat added; lane 3, transcription with 50 ng of Tat; lane 4, transcription in the absence of Tat and with 100 ng NIPP1; lane 5, transcription in the presence of 50 ng of Tat and 100 ng NIPP1. Transcription products were resolved on 5 % Urea-PAGE, exposed to the PhosphoImager screen and quantified.