Proteasome inhibitors suppress Btk transcription, a phenomenon requiring de novo protein synthesis. (A) A20 cells were treated with CHX (10 μg/mL) an hour prior to the treatment with epoxomicin (10 nM) or MG132 as indicated. Cell lysates were subjected to Western blot analysis. Total protein ubiquitin level was used for assessing the activity of the proteasome inhibitors, and ERK1 and ERK2 served as internal controls. An unknown 50-kD protein also served as loading control. (B) A20 cells were treated with CHX and MG132 as in panel A. Cytosolic and nuclear proteins were obtained using an extraction kit from Pierce, and proteins were processed for Western blot analysis as indicated in the figure. (According to the company's instruction, the cytosolic proteins were finally dissolved in 211 μL buffer, whereas nuclear proteins were dissolved in 100 μL buffer.)