Reduction of CDK2 activity in SCD PBMCs. (A) CDK2 activity is reduced in SCD PBMCs. PBMCs were obtained from 3 SCD patients and 3 control subjects and activated with PHA and IL-2. Cells were lysed, and CDK2 was immunoprecipitated using anti-CDK2 antibodies from lysates that were equalized by protein. Kinase assays were performed using histone H1 as a substrate. Upper panels show a representative immunoblot of CDK2, a radioactive image of phosphorylated histone H1, and a Coomassie-stained image of histone H1. Lower panel shows quantification from 3 independent experiments. The means ± SD and P values calculated using Student t test are shown. (B) CDK2 knockdown inhibits HIV-1 infection in THP-1 cells. THP-1 cells were stably transduced with lentiviruses expressing CDK2-targeting shRNA or control shRNA and then infected with HIV-1-LUC-G virus. Left panel shows luciferase activity measured at 48 hours PI. Right panel shows expression of CDK2 mRNA determined by RT-PCR with 18S RNA as an internal reference for ΔΔCt analysis. The means ± SD are shown for 3 CDK2 KD and 3 control shRNA clones. (C) CDK2 activity is reduced in hemin-treated THP-1 cells. THP-1 cells treated with 75 μM hemin and control cells were lysed, and CDK2 was immunoprecipitated using anti-CDK2 antibodies. Kinase assays were performed using histone H1 as a substrate. Lower panel shows quantification from 3 independent experiments. P value was calculated using Student t test. (D) Hemin treatment reduces CDK2 mRNA expression. THP-1 cells were treated with 75 μM hemin for 24 hours. RNA was extracted, reverse transcribed, and analyzed by RT-PCR using primers for CDK2 and 18S rRNA as an internal reference for ΔΔCt analysis. The means ± SD and P values calculated using Student t test are shown for 3 independent samples. N/s abs, nonspecific antibodies.