Fig. 7.

Functional consequences of ILK mutant alleles. a Western blot analysis of β-catenin and AKT Ser473 phosphorylation in HUVEC after siRNA-mediated knockdown of endogenous ILK and expression of siRNA-resistant wild-type (WT) ILK and variant versions, as indicated. Quantitation provided in Supplementary Fig. 5A. b Paxillin (red) and DAPI (nuclei, blue) stained HUVECs showing the changes in cell morphology and focal adhesion formation (high-magnification insets) after knockdown (KD) of endogenous ILK expression and transfection with either GFP alone (GFP), a GFP fusion with wild-type (WT) ILK, or GFP fusions with L53M, R211C, and R317Q ILK. ILK KD-induced cell spreading and focal adhesion defects are reverted by expression of WT GFP-ILK but none of the three mutant constructs. Scale bar, 20 μm. c Immunoprecipitation (IP) and Western blot analysis of wild-type (WT) or mutant GFP-ILK and associated PINCH and α-parvin proteins. White arrows indicate GFP-ILK and black arrows endogenous ILK. d Confocal images of p-AKT (Ser473) (green) and isolectin B4 (white) staining in control and Ilk^iECKO P6 retina sections. Presence (control) or absence (Ilk^iECKO) of p-AKT in endothelial nuclei (red arrows) in images with or without DAPI signal (blue). Scale bar, 10 μm. e qPCR analysis of AXIN2 expression in HUVEC after siRNA knockdown of endogenous ILK and expression of siRNA-resistant wild-type (WT) or variant ILK proteins, as indicated. Error bars, s.e.m. p values (**p < 0.01, *p < 0.05, ns not significant), Student’s t test (n = 6). Source data are provided as a Source Data file