Upstream signaling responsible for eIF2-α phosphorylation: A role for Nck1. A-B: MDA-MB-231 cells and BT474 cells were treated with vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 μM), lapatinib (Lap, 2 μM) or the combination (Combo) for 3 h, then harvested for immunoblotting assays as described in Materials and Methods. Membranes were probed with the indicated antibodies. C: MDA-MB-231 cells (5 x 105) were transfected with plasmids to express either GFP-Nck1 or GFP-Nck2 as described. After an additional 24 h, cells were treated with the combination of OSU-03012 and lapatinib as indicated for 24 h (upper graphs) or 3 h (lower panels), and then either plated onto 6-well dishes and allowed to form colonies (graphs represent percent control) or harvested for immunoblotting assays and probed with the indicated antibodies. * indicates a p<0.05.