Journal: Journal of Cell Science

Article Title: Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell–cell and cell–substrate adhesion

doi: 10.1242/jcs.140475

Figure Lengend Snippet: LPP-knockdown cells show diminished E-cadherin-dependent adhesion. (A) MDCK control and two separate LPP-knockdown cell lines were cultured on glass coverslips for 3 days and either extracted (right panels) or not (left panels) with CSK buffer before fixation and immunofluorescence staining for E-cadherin. Scale bar: 20 µm. (B) Representative line scan analysis of images as shown in C demonstrates little loss of E-cadherin fluorescence in control cells after CSK extraction (top panels). By contrast, there is greater loss of signal from E-cadherin fluorescence in LPP-knockdown cells after CSK extraction (right two bottom panels) compared with unextracted cells (left two bottom panels). (C) Schematic of modified assay to measure E-cadherin-dependent adhesion. MDCK and LPP-knockdown cells were separately labeled with fluorescent dyes, mixed and incubated for 60 minutes on E-cadherin- or fibronectin-coated plates. Wells were washed to remove loosely adhering cells and the relative fluorescent signals from MDCK and LPP-knockdown cells were compared with unwashed wells. (D) MDCK control cells adhered twice as well as LPP-knockdown cells to E-cadherin-coated wells; there was no difference in adhesion to fibronectin in the control versus knockdown cells. Values are means ± s.d.; *P<0.005 by unpaired Student's t-test.

Article Snippet: To coat wells with substrate, 75 μl/well of 10 μg/ml E-cadherin (R&D Systems recombinant human E-cadherin FC Chimera, Minneapolis, MN) or fibronectin (Life Technologies) was added to a 96-well plate and incubated overnight at 4°C; 4 hours before use, wells were blocked with 10% BSA.

Techniques: Cell Culture, Immunofluorescence, Staining, Fluorescence, Extraction, Modification, Labeling, Incubation