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Fig. 4. ILK silencing suppresses fibronectin fibrillogenesis. (A) Formation of extracellular fibronectin fibrils was determined by immunostaining of control or ILK siRNA-transfected cells. 24 hours after the second transfection, cells were plated for 32 hours on uncoated glass coverslips before being fixed and stained for fibronectin without permeabilization. (middle) Cell nuclei stained with DAPI (scale bar, 20 µm). (bottom) Merged images of cells fixed after 2.5 hours of adhesion to uncoated coverslips and permeabilized before co-staining for fibronectin (red), the 
5 subunit of 5ß1 (green) and DAPI (blue). Scale bar, 20 µm. Immunostaining was performed at early times after plating to limit the extracellular accumulation of fibronectin fibrils. (B) 48 hours after the second transfection, cell-associated fibronectin was analysed by western blotting. The same membrane was reblotted for ILK expression to determine the efficiency of ILK silencing in the experiment. Total ERK1/2 levels were analysed as a control for equal protein loading. (C) Northern-blot analysis of fibronectin and ILK mRNA expression in control and ILK siRNA. A probe corresponding to 36B4 (GenBank accession number M17885) was used to control for equal RNA loading. (D) Fibronectin in concentrated 24-hour conditioned medium (corresponding to 250 µl) from control or ILK siRNA-transfected cultures was detected by western blotting. Values below correspond to the fold increase in soluble fibronectin secreted from ILK siRNA-transfected cells relative to control cells (mean ± s.e.m. from three independent experiments).
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