Figure 5.

ERK/MAPK signaling leads to the sequestration of Bim-EL from the mitochondria to prevent anoikis. (a) Cells were plated on poly-HEMA-coated plates with DMSO or the indicated dose of U0126 or PD0325901 for 6 h. Lysates were prepared and normalized, and protein levels were analyzed via western blotting. (b) Cells were plated in detachment and treated with DMSO or U0126 (10 μM) for 3 h. Cell lysates were prepared, and immunoprecipitation was performed with a Beclin-1 antibody. Western blotting analysis was utilized to identify interacting proteins and confirm equivalent protein content across samples. (c) Cells were plated in detachment and treated with DMSO or U0126 (10 μM) for 3 h. Cell lysates were prepared and normalized, and immunoprecipitation was performed with a Bim antibody. Interacting proteins were identified via immunoblotting, and equivalent protein content across samples was confirmed. (d and e) Cells were plated on poly-HEMA-coated plates and treated with either DMSO or U0126 (10 μM) and 20 μM z-VAD-fmk for 24 h. Cells were fixed and stained with Mito-Tracker Red (200 nM), DAPI (5 μg/ml), and Bim-EL and imaged using an Applied Precision DeltaVision OMX fluorescent microscope. Co-localization was measured using the Applied Precision softWoRx software. Error bars represent S.E.M.