Figure 5
From: Reactivation of latent HIV-1 provirus via targeting protein phosphatase-1
Effect of SMAPP1 on CDK9 phosphorylation. a SMAPP1 induces CDK9 Ser90 phosphorylation. 293T cells were transfected with vectors expressing Flag-CDK9 WT (lanes 1–2) or Flag-CDK9 S90A mutant (lanes 3–4) and after 48 h in culture treated with 10 μM SMAPP1 for 24 h. CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies and analyzed by immunoblotting with anti-CDK9 antibodies and Ser90 phospho-specific antibodies. b Quantification of four independent experiments similar to the one shown in a. Asterisk shows p value ≤0.05. c Effect of SMAPP1 on CDK9 Ser175 and Thr186 phosphorylation. 293T cells were transfected with vectors expressing Flag-CDK9 WT (lanes 2–4) or Flag-CDK9 S90A mutant (lanes 5–6) or Flag-CDK9 S175A (lanes 7–8) and after 48 h in culture treated where indicated with 10 μM SMAPP1 for 24 h or 0.1 μM okadaic acid for 2 h. Lane 1 mock transfection. CDK9 was immunoprecipitated from cellular lysates with anti-Flag antibodies and analyzed by immunoblotting with anti-CDK9 antibodies, Ser175 phospho-specific and Thr186 phospho-specific antibodies. d, e Quantification of two independent experiments. Asterisk shows p value ≤0.05 and double asterisk shows p value ≤0.01 between the indicated bars.