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Figure 1. Effects of TNF on the molecular interaction between KIF5B and ß-tubulin-III, as determined by lifetime-based FRET analysis. Neurons were coimmunolabeled with antibodies directed against KIF5B marked with FITC and antibodies directed against ß-tubulin-III marked with Cy3. Lifetime images were acquired of KIF5B-FITC. Lifetimes from 1.0 to 1.9 ns were coded in red, representing expected signals from FRET. Lifetimes from 1.9 to 4.0 ns were coded in green, representing the expected lifetime of KIF5B-FITC in the absence of acceptor. In untreated neurons, direct association between ß-tubulin-III and KIF5B was observed as demonstrated by lifetime values coded in red. After treatment with TNF for 20 min, the conformation of the KIF5B-ß-tubulin-III complex changed in neurites as demonstrated by lifetime values coded in green. Specifically, TNF induced a breakage of the normal KIF5B-ß-tubulin-III complex in neurites, but not in cell bodies. Scale bars: 20 µm.
