32D/Bcr–Abl cells do not require NF-κB for survival in the absence of IL-3. (A) Production of 32D/Bcr–Abl/IκBα-SR cells. 32D/p185 and 32D/p210 cells were infected with a retrovirus expressing IκBα-SR. Mass population and clonal stable cell lines were obtained by G418 selection. Cell extracts were prepared from 32D, 32D/p185, 32D/p210, 32D/p185/IκBα-SR, and 32D/p210/IκBα-SR cells by lysing equal cell numbers in protein SDS sample buffer. Extracts were analyzed by Western blot analysis with an Abl monoclonal antibody and a carboxy-terminal IκBα polyclonal antibody. Mobility of p210, p185, endogenous c-Abl, IκBα-SR, and endogenous IκBα are indicated with arrows; (n.s.) indicates a nonspecific band. (B) 32D/Bcr–Abl cells expressing the IκBα-SR no longer contain NF-κB DNA binding. EMSAs were performed with nuclear extracts from 32D, 32D/p185, 32D/p210, 32D/p185/IκBα-SR, and 32D/p210/IκBα-SR stable cell lines and an oligonucleotide probe containing an NF–κB binding site. NF-κB complexes are indicated with arrows. (C) 32D/Bcr–Abl/IκBα-SR cells undergo apoptosis in response to TNFα. 32D (open bars), 32D/p210 (hatched bars), 32D/p210/IκBα–SR–mp (vertical bars), and 32D/p210/IκBα-SR clonal cell line H3 (solid bars) were treated with TNFα for 24 hr. Cells were stained with propidium iodide at 0 hr and 24 hr post-TNFα treatment and DNA content was analyzed by flow cytometry. Cells within the sub-G1 peak were scored as apoptotic. (D) 32D/p185/IκBα-SR cells survive in the absence of IL-3. 32D and 32D/p185/IκBα-SR cells were incubated in the presence or absence of IL-3 and cell viability was monitored by trypan blue exclusion 24 hr and 48 hr post-IL-3 withdrawal.