Increased transcriptional activity of PU.1 in cells expressing p15Ink4b. (A) Cell lysates from RAW264.7 cells expressing empty vector (Mig0) or p15Ink4b (MigDDp15) and treated with LPS (1 μg/mL) for the indicated periods were analyzed by Western blot with Abs against the indicated phosphorylated IκBα (Ser-32) or NF-κBp65 (Ser536) and the total IκBα, NFκBp65, and PU.1 (Cell Signaling Technology) proteins. The representative results shown are from the same blot that was stripped and reprobed with anti-actin. (B) Binding of PU.1 to the CD80 or CD86 promoter in RAW264.7 cells was evaluated by ChIP assay using qPCR. Results were normalized to histone H3 ChIPs and are plotted as the -fold enrichment over the normal IgGs. (C) Transcriptional activity of PU.1 was evaluated by dual luciferase reporter assay in RAW264.7 cells transfected with the PU.1-dependent luciferase reporter pPU.1-Luc construct and the Renilla luciferase pCMV-RL construct. Twenty-four hours after transfection, cells were either treated or untreated with LPS (1 μg/mL) for another 12 hours, then lysed and analyzed for luciferase activity by luminometry. Data are shown as means ± SD from 2 independent experiments. *P < .05.