Fig. 2: NET4 proteins co-localize, interact with each other and themselves. | Nature Communications

Fig. 2: NET4 proteins co-localize, interact with each other and themselves.

From: NET4 and RabG3 link actin to the tonoplast and facilitate cytoskeletal remodelling during stomatal immunity

Fig. 2: NET4 proteins co-localize, interact with each other and themselves.The alternative text for this image may have been generated using AI.

a Confocal micrographs of N. benthamiana leaf epidermal cells transiently co-expressing NET4A-GFP and NET4B-RFP. Shown are single plane confocal images capturing GFP fluorescence (green), RFP fluorescence (red) and overlaid signals (yellow); scale bars = 20 μm. The experiment was repeated at least thrice with similar results. b Yeast-two-hybrid analysis of the indicated constructs indicate homo- and heterodimer interactions of NET4A and NET4B (EV = empty vector). The experiment was repeated at least thrice with similar results. c Co-immunoprecipitation (Co-IP) assay of NET4A-GFP with NET4B-RFP. Fusion proteins were co-expressed in N. benthamiana, immunoprecipitated (IP) using GFP-trap beads and detected using fluorophore fusion specific antibodies (GFP and RFP). 1% of the input is shown as loading control. Blots are representative of two independent experiments. d FRET-FLIM analysis of NET4A-GFP and NET4B-RFP interaction. FLIM (TCSPC) confocal images of N. benthamiana leaf epidermal cells co-expressing both NET4 proteins. Data shown using lifetime LUT. e Graph of fluorescence lifetime values of donor alone and donor + acceptor. The significant reduction in lifetime of the donor in the presence of the acceptor indicates energy transfer and a physical interaction; n = 10, p < 0.0001 Mann Whitney t-test; errors bars represent SD. The experiment was repeated at least twice with similar results.

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