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Figure 1. Membrane-targeted ILK. A) Schematic representation of membrane-targeted integrin-linked kinase (ILK-GFP-F). The C-terminal of ILK was fused to a green fluorescent protein (GFP) fitted with the farnesylation sequence of H-Ras. B) ILK-GFP and ILK-GFP-F expression in CCL39 fibroblasts was monitored by Western blotting using an anti-ILK Ab (DI). C) Exponentially growing ILK-GFP and ILK-GFP-F cells were subjected to hypotonic lysis and isolation of the membrane (M) and soluble (S) fractions was performed. Ten micrograms of protein were separated by SDS-PAGE and analyzed by Western blotting using antitransferrin receptor as a marker for the membrane and anticaspase 9 as a marker for the cytosol. Results are representative of 2 independent experiments. Chimeric ILK proteins were detected with anti-V5 Ab, and endogenous ILK was detected with the DI anti-ILK Ab. D) The mean surface area (±SEM, n>60) was determined on F-actin-stained cells 20 h after plating on fibronectin-coated coverslips. E) CCL39-derived cells expressing EGFP-F, ILK-GFP, or ILK-GFP-F were plated on fibronectin-coated coverslips for 20 h then fixed, permeabilized and stained for paxillin. Digital overlay of images was performed using MetaMorph software. Scale bar = 20 µm.
