CD257 enhances survival of CD3⁻CD5⁺ CLL cells from TCL1-Tg mice and activates NF-κB. (A) CD3⁻CD5⁺ CLL cells from TCL1-Tg mice were cultured in absence (top line) or presence (bottom line) of 200 ng/mL rhCD257 and apoptosis was determined by TUNEL assay at different time points as indicated (n = 3 independent experiments). One representative assay is shown for each experiment. (B) Statistical analysis of data obtained in panel A. Error bars represent SD of 3 independent experiments. (C) Immunoblot from nuclear extracts of cells treated for different times with 200 ng/mL rhCD257, or left untreated, using antibodies to detect activation of the classical (p65) or alternative (RelB, p52) NF-κB pathway. HDAC1 served as a loading control. Shown is a representative blot (n = 3 independent experiments). (D) Immunoblot of cytosolic extracts of the same cells used in panel C immunoblotted for the antibodies indicated. Tubulin was used as a loading control. Shown is a representative blot (n = 3 independent experiments).