Proteasome and/or NF-κB inhibitors regulate Btk transcription. (A) A20 cells were treated with or without MG132 for 16 hours and total RNA was isolated and subjected to RT-PCR. Relative amount of Btk mRNA levels is demonstrated at the bottom of the figure. Data are representative of 3 independent experiments. (B) Ramos cells were treated with or without MG132 for 16 hours. The amount of Btk mRNA was evaluated by quantitative RT-PCR (triplicate samples were run). Each sample was normalized using 18S rRNA as a control. (C) Schematic representation showing the structure of the Btk promoter. Depicted here are some transcription factors and coactivators (OCT1, PU.1, and sp1/sp3) that have been demonstrated to bind to the Btk promoter. Btk promoter reporter constructs (500-Btk and 1000-Btk) and 2 putative NF-κB–binding sites are also indicated. (D) The 500-Btk and 1000-Btk promoter constructs were introduced into A20 cells, and cells were treated with or without MG132 for 16 hours. Luciferase activity was measured as described in “Methods”; relative levels of luciferase activity are shown. Data are representative of 3 independent experiments. (E) A20 cells were treated with bortezomib (20 nM) or Bay 11-7085 (5 μM) for 16 hours. Cell lysates were subjected to Western blot analysis as indicated. (F) NMRI mice were transfected with 20 μg of the 1000-Btk promoter reporter construct using the hydrodynamic procedure. In addition, the mouse in the middle received 1 mg/kg bortezomib and the mouse to the right received 1 mg/kg LPS. In vivo biophotonic imaging was performed using the IVIS imaging system as described in “Methods.” Data are representative of 3 independent experiments.