Nck1, but not Nck2 expression ablates the increase in cell death induced by OSU-03012 and lapatinib. A-D: MDA-MB-231 cells (A, B), or BT474 cells (C, D) (5 x 105) were co-transfected with either wild-type eIF2-α and the two Nck isoforms (A, C), or the S51A phospho-mutant and the two Nck isoforms (B, D). Cells were allowed to incubate for 24 h to induce ectopic expression, and then treated with either vehicle (indicated with a -), or the combination of OSU-03012 and lapatinib (2 μM each, indicated with a +). Cells were either harvested after a 3 h treatment for western blotting as described in Materials and Methods (bottom panels), or plated singly onto 6-well plates to assay for clonogenic capacity (upper graphs). Cells were treated with OSU-03012 (2 μM) and lapatinib (2 μM) for 24 h, and then allowed to form colonies for 10-14 days. Total colony counts were graphed. * denotes a p-value of <0.05.