Figure 3

Association of Dsg3 with Ezrin. (a) In situ PLA assay in A431 cells showed increased signals of Dsg3/Ezrin interaction (red dots) in cells with overexpression of Dsg3 (polyclone:D3; monoclones: C2, C7 and C11) compared with Vect or positive (+) and negative (−) controls (mean±s.d., **P<0.01). Here, the negative control was Dsg3/myc-tag in A431-Vect cells and the positive was Dsg3/myc-tag in A431-D3 cells. (b) Confocal analysis of FRET by acceptor photobleaching: the representative images are shown and the enlarged region 1 (Reg-1) for each channel is displayed at the bottom panels. The bleach of acceptor was 61.24% (average of Reg-1 and -2) and the enhanced fluorescent intensity in region 1 and 2 compared with internal and photo-damage regions is shown below the images. Among 89 regions comprising both membrane projections and intercellular junctions, 13 displayed positive FRET efficiency (14.6%) above thresholding of 5%. The FRET efficiency was calculated by a formula (Materials and methods), and is shown at the bottom of (b; Negative: n=16 and Positive: n=13, mean±s.d., ***P<0.001). Scale bars, 10 μm in (a), 20 μm in (b). (c, d) Co-immunoprecipitation (co-IP) in both A431-Vect and -D3 cells. The representatives from at least six independent co-IPs shown in (c) and from at least two independent co-IPs in both polyclonal and monoclonal cell lines are shown in (d), demonstrating that the association exists for both endogenous and exogenous Dsg3 and is in a Dsg3 dose-dependent manner. Scale bars, 10 μm.