Fluorescence anisotropy measurements were performed as described recently (Brooks et al., 2014 (link)). Briefly, MCF-7 cells were transiently transfected with mCherry or mRPTPα-mCherry or cotransfected with mRPTPα-mCherry and mEcad-GFP (pLL5.0, mE-cadGFP). Cells were imaged live at 37°C by confocal microscopy, and images were acquired on a LSM710 Zeiss confocal microscope equipped with a Plan-Apochromat 40×/1.4 NA oil-immersion objective (Zeiss, Jena, Germany). Images were acquired by using a 561-nm laser excitation line and collecting the corresponding emissions in the range of 580–620 nm using band-pass emission filters. Fluorescence emission was split into the components parallel (Ipar) and perpendicular (Iper) to the plane of excitation by a polarized beam splitter. The fluorescence anisotropy was determined by measuring average values of Ipar and Iper on regions of interest at the cell–cell junctions in Image J and using the following equation:
where the correction factor G was estimated using r0 = 0.35 of soluble mCherry as reference (Bader et al., 2011 (link); Lindenburg et al., 2013 (link)). Data presented are mean r values calculated across the different images (∼50 cells per condition) and their SEs.
where the correction factor G was estimated using r0 = 0.35 of soluble mCherry as reference (Bader et al., 2011 (link); Lindenburg et al., 2013 (link)). Data presented are mean r values calculated across the different images (∼50 cells per condition) and their SEs.