Expression of Tcl1 and Atm activates the NF-κB pathway. (A) HEK293 cells were transfected with indicated plasmids. Forty-eight hours later, cells were treated with MG132, 10μM for 3 hours, and then lysates immunoprecipitated with anti-V5. The precipitates were analyzed by Western blot with anti-HA or anti-IκBα. Input: lysate expression of Tcl1 and Atm. (B) HEK293 cells were mock-transfected or transfected with TCL1 and ATM plasmids separately or together (4 μg each plasmid). Forty-eight hours later, cells were treated with hydroxyurea for 3 hours, fractionated into cytosolic and nuclear fractions, and analyzed by Western blot with the indicated antibodies. (C) mRNA levels of EGR1 were measured by quantitative RT-PCR. HEK293 cells were mock-transfected or transfected with TCL1 and ATM plasmids, separately or together for 48 hours, and then treated with hydroxyurea, 50mM for 3 hours). Fold changes of EGR1 were calculated using the 2^−ΔCt method. GAPDH mRNA levels were used as an internal normalization control. Samples transfected with TCL1 and ATM have been normalized to mock-transfected sample. (D) HEK293 cells were transfected with TCL1 and ATM (4 μg for each plasmid), treated with hydroxyurea for 3 hours and with MG132, 10μM 6 hours, and protein lysates run on gel. (E) Tcl1 activates NF-κB–dependent transcription synergistically with Atm. HEK-293 cells were cotransfected with 50 ng of pNF-κB-Luc reporter and 50 ng of pRL-TK Renilla reporter constructs. In addition, 0.75 μg of CMV5-empty vector, 0.75 μg of pcDNA 3.1 empty vector, 0.75 μg of CMV5-TCL1 WT, and 0.75 μg of pcDNA 3.1 empty vector constructs were used; 5 ng of pFC-MEKK was added where indicated. Cells were treated with 10μM Kudos for 5 hours, where indicated. Data are representative of 3 independent experiments. (C,E) Data are mean ± SEM of 3 independent experiments, and each is measured in triplicate. *P < .05.