Fig. 1

α2β1 integrins are required for stable adherens junctions. a Confocal image of normal human keratinocytes (WT) monolayers in 2-mM Ca²⁺ fixed and stained for F-actin and α2 integrin. Scale bar 10μm. b Western blot of lysates from WT, control shRNA (Con) and α2 knockdown shRNAs (KD1 and KD2) keratinocytes probed for α2 integrin and HSC70 loading control. c Confocal image of control and α2KD keratinocytes stained for E-cadherin (E-cad), F-actin and α2 integrin. Scale bars 10μm. d Line graph and quantification of E-cad intensity distribution across junctions, and quantification of peak intensity at junctions from images as in c. Data are from at least 30 images per condition over 3 independent experiments. e Line graph and quantification of F-actin intensity distribution across junctions from images as in c. Data are from at least 30 images per condition over 3 independent experiments. f Representative western blot of control and α2KD cells subjected to surface biotinylation and stripping at 30/60 min followed by Streptavidin IP and probing for E-cadherin (top blots). E-cadherin input shown on bottom blot. Graph shows quantification of internalised E-cad from 4 independent experiments (mean+/−SEM). g Lysates from control and α2KD cells immunoprecipitated with β-catenin antibodies and complexes probed for β-catenin (β-cat) and E-cadherin. h WT cell lysates +/−2mM Ca²⁺ immunoprecipitated with α2 antibodies or control IgG and complexes probed with E-cadherin, β-catenin or α2 integrin antibodies. i GFP trap of lysates from α2KD cells expressing GFP or α2-GFP in 2-mM Ca²⁺ treated with DMSO (−) or BTT3033 (BTT; +) and probed for β-catenin and GFP. j X,Z reconstructions from structured illumination microscopy (SIM) images of WT junctions in 2-mM Ca²⁺ stained for α2 integrin and β-catenin. Scale bar 1μm. ***p<0.001, **p<0.01