Supplementary Figure 1: Characterization of α5/2 chimera endothelial cells.

(a) BAECs expressing human integrin wild type α5 or the α5/2 chimera were stained with mAb16 that recognizes human integrin α5 extracellular domain. (b) Wild type α5 or the α5/2 chimera cells were lysed and immunoprecipitated with mAb16 to isolate exogenous human integrin α proteins. Western blots with antibodies against the α5 and α2 cytoplasmic tails confirm the sequences (upper and middle panels), and show similar pairing with the β1 subunit (lower panel). (c,d) Wild type α5 and α5/2 chimera cells spreading on fibronectin. BAECs expressing wild type integrin α5 or the chimera were detached and replated on dishes coated with fibronectin (10 μg/ml) for the indicated times. The cells were either fixed and stained with wheat germ agglutinin (c) or lysed and subjected to immunoblotting for FAK phosphorylation (Y397) (d). (e) Wild type α5 or α5/2 chimera cells were plated on fibronectin and grown to monolayer and then kept in low-serum media (1% FBS) for two days to induce fibronectin fibrillogenesis. The cells were fixed and stained for fibronectin. For each condition, n = 30 images pooled across three independent experiments were averaged. The box plot shows the median, with upper and lower percentiles, and the bars show maxima and minima values. (f) Wild type α5 or α5/2 chimera cells were plated on fibronectin and sheared for 36 hrs (20dynes/cm²). Cells were stained with phalloidin and Hoechst and alignment in the direction of flow was quantified (±30°). Scale bars: 50 μm. n = 10 images (60-100 cells/field) were used for quantification for each condition. Error bars are SEM.