TCL1 inhibits ERK activation. (A) Jurkat-C (gray box) and Jurkat-TCL1 (black box) were either treated or untreated with PMA in presence or absence of U0126, added 1 hour before and during the stimulation period. Cells were collected after 24 hours, stained with PI, and analyzed for cell cycle distribution (S phase) using flow cytometry. Three additional experiments gave similar results. Error bars represent SD. (B) Photomicroscopy of Jurkat-C and Jurkat-TCL1, treated or untreated with PMA for 3 days in presence or absence of U0126. Two additional experiments gave similar results. (C) Jurkat-C and Jurkat-TCL1 were stimulated with PMA at different times, as indicated in the figure. ERK phosphorylation (p-ERK) and TCL1 expression were determined by Western blotting. Total ERK expression was used as a loading control. A representative experiment of 5 performed is shown. Two polyclonal populations from independent transfections were tested with similar results (data not shown). (D) Jurkat-C and Jurkat-TCL1 were stimulated with UCHT1 at different times, as indicated in the figure. ERK phosphorylation (p-ERK), AKT phosphorylation (p-AKT), and TCL1 expression were determined by Western blotting. β-Actin expression was used as a loading control. A representative experiment of 4 performed is shown. (E) Jurkat-C and Jurkat-TCL1 were stimulated with UCHT1 for 10 minutes, and ERK was analyzed by immunofluorescence using an anti-ERK (FITC). Representative images of 3 independent experiments are shown. (F) Jurkat-C and Jurkat-TCL1 were stimulated with UCHT1 in presence or absence of rottlerin (10 μM) or Gö6850 (1 μM), added 1 hour before and during the stimulation period. ERK phosphorylation was determined by Western blotting. After quantification, the p-ERK/β-actin ratio was determined as indicated at the bottom. Vertical lines have been inserted to indicate a repositioned gel lane. A representative experiment of 2 performed is shown.