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doi:10.1016/S0092-8674(00)80595-4. and short-lived ERK activity, and insulin-like growth factor-I stimulated strong long-term Akt responses but negligible ERK signaling. To address potential interactions between signaling pathways, we employed specific small-molecule inhibitors. In cells incubated with EGF or platelet-derived growth factor-AA, Raf activation and the subsequent stimulation of ERK reduced Akt signaling, whereas Mek inhibition, which blocked ERK activation, enhanced Akt and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly Balsalazide disodium in Balsalazide disodium strength and duration and demonstrate in living cells the dramatic effects of cross talk from Raf and Mek to PI 3-kinase and Akt. Our data further indicate NESP how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways. coordinates at could be propagated across the entire image stack using the Multi Measure tool. The location of each nucleus from the green channel image (FoxO1-clover) was used to quantify nuclear intensity of the red channel image (mKate2-ERK2). Nuclear intensities for FoxO1-clover and mKate2-ERK2 in each cell were normalized to the value recorded at and then scaled based on the average maximal reporter translocation after incubation of cells with EGF (SFM = 0%, peak EGF = 100%). To measure the subcellular distribution of FoxO1-clover in C3H10T1/2 cells the nuclei of individual cells were manually tracked across frames using the mTrackJ plug-in (31). Cells that died, divided, or migrated out Balsalazide disodium of frame were excluded from analysis. Imaging protocols. HeLa cells were grown in DMEM containing 10% FBS for 48 h to allow full cell attachment. After two washes with DMEM, cells were incubated in serum-free Fluorobrite imaging medium for 2 h. Growth factors and/or inhibitors were then added, and images were collected every 2.5 min for 90 min. Growth factors included R3-IGF-I Balsalazide disodium (0C250 pM), EGF (0C2.1 nM), HGF (0C1.7 nM), insulin (1 nM), and TGF- (1.67 nM). For inhibitor studies, HeLa cells were incubated in SFM for 2 h followed by addition of growth factor [EGF (2.1 nM), R3-IGF-I (250 Balsalazide disodium pM), or HGF (1.72 nM)], with or without PI-103 (500 nM), U-0126 (10 M), trametinib (500 nM), or MK-2206 (1 M). C3H10T1/2 cells were incubated in SFM for 90 min, followed by the addition of growth factors [EGF (2.1 nM) or PDGF-AA (1.4 nM)] with or without PI-103 (500 nM), U-0126 (10 M), or PLX-4720 (10 M). Images from C3H10T1/2 cells were collected every 2 min for 90 min. For all imaging studies a minimum of three independent experiments was performed. Protein extraction and immunoblotting. Whole cell protein lysates were collected after washing cells two times with cold PBS followed by the addition of extraction buffer containing protease and phosphatase inhibitors. Protein aliquots (12.5 g/lane) were separated by SDS-PAGE (12% separating gels) followed by transfer to Immobilon-FL membranes, blocking with 50% AquaBlock solution, and sequential incubation of membranes with primary and secondary antibodies, as described (34). Primary antibodies were added at 1:1,000 dilutions for 16 h at 4C and secondary antibodies at 1:5,000 dilution for 90 min at 20C. Images were captured using the LiCoR Odyssey and version 3.0 analysis software (Lincoln, NE). For analysis by protein array, whole cell protein lysates were collected from HeLa cells after addition of the provided cell lysis buffer (Pathscan Antibody Array Kit; Cell Signaling) supplemented with protease inhibitors. Protein aliquots (75 g/well) from cells incubated with SFM or EGF (2.1 nM) for 15 min PI-103 (500 nM) and/or U-0126 (10 M) were added to each slide well and incubated at 4C for 16 h. Slides were washed four times with the provided wash buffer and then incubated for 1 h with the detection antibody cocktail at 20C. Following four.