TCL1 inhibits NF-κB activation. (A) Jurkat-C and Jurkat-TCL1 were stimulated with PMA or UCHT1 at different times indicated in the figure. IκBα (p-IκBα, top) and PKCθ (p-PKCθ, middle) phosphorylation was determined by Western blotting. β-Actin expression was used as a loading control (bottom). A representative experiment of the 3 performed is shown. (B) Band shift assay of nuclear extracts from Jurkat-C and Jurkat-TCL1. Cells were either untreated or stimulated for 30 and 60 minutes with PMA in presence or absence of Bay 11-7085 as indicated in the figure. A constitutive DNA binding of NF-κB with the same intensity in Jurkat-C and Jurkat-TCL1 is indicated by a gray arrow. Asterisk (*) indicates lane containing a 100-fold excess of cold KBF1 oligonucleotide competitor. (C) Jurkat-C (▒) and Jurkat-TCL1 (■) were seeded at 0.5 × 106 cells/mL, and treated (□) or untreated (plain box) with PMA (20 ng/mL) in presence or absence of Bay 11-7085 added 1 hour before and during the stimulation period at indicated concentrations. The viable cell number was determined at 48 hours, using a cell viability analyzer (Vi-cell XR; Beckman). Data are expressed as mean (± SE) of duplicates samples. One additional experiment gave similar results. (D) Photomicroscopy of Jurkat-C and Jurkat-TCL1, treated or untreated with PMA for 3 days in presence or absence of Bay 11-7085 at indicated concentrations. Two additional experiments gave similar results.
