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Fig. 5. FLIM analysis of BACE phosphorylation. N2a cells were transfected with BACE-GFP, treated with 20 nM OA and stained with an antibody specific to phosphorylated serine residues for the analysis. Negative controls were only transfected with BACE-GFP and treated with OA (A,B). The intensity image shows the standard immunostaining pattern for BACE (A,C). The color-coded FLIM image shows the lifetimes (in picoseconds) of GFP in the presence of acceptor Cy3. The lifetime reflects the proximity between BACE-C-terminus and phosphoserine residues, demonstrated in pseudocolor. The FLIM image suggests that there is a close proximity between the BACE C-terminus and phosphoserine residues not only in juxtanuclear compartments (TGN), but throughout the cell (D). The chart shows mean changes in lifetime and statistically shorter lifetimes in the presence of Cy3-labeled endogenous phosphoserine (P<0.001).





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