Figure 3

Effects of different liprin-α1 constructs on the subcellular localization of ERC1.

(a) Laser scanning confocal microscope images of MDA-231 cells plated on fibronectin. Left is a merge of the confocal image (green) and phase contrast to show the shape of the cells transfected with the indicated GFP-tagged liprin-α1 construct. Graph on the right: quantification of transfected cells with diffuse (GFP-positive) signal or with signal concentrated at the cell edge (n = 31–49 cells from 3 independent experiments). ***P < 0.0001 by the χ² test. (b) Cells were seeded on fibronectin-coated coverslips, fixed and processed for immunofluorescence to reveal the indicated endogenous proteins. Images were acquired at UltraViewer spinning disk confocal microscope. Scale bar 20 μm. (c) TIRF microscopy of cells cotransfected with the indicated GFP-tagged liprin-α1 constructs (green) and mCherry-ERC1 (red). The upper row shows the merge of green (second row) and red (bottom row) channels. Scale bar, 20 μm. (d) TIRF microscopy of cells transfected with the indicated GFP-tagged liprin-α1 constructs (green) and siRNAs, immunostained to detect endogenous ERC1 (red). From left: the first column shows the merged TIRF images at the bottom of the cells (90 nm); the second and third rows show the distributions of the transfected liprin-α1 constructs and of endogenous ERC1, respectively; the last two columns to the right show 4-fold enlargements of the protrusions highlighted in the first column. Scale bars, 20 μm.