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Figure 2


Fig. 2. Podoplanin binds to ERM proteins through a cluster of basic amino acids within its cytoplasmic tail. (A) Association of ezrin and moesin with podoplanin CT. GST and GST fusion proteins bound to Sepharose beads were incubated with purified recombinant full-length ezrin or moesin or their N-ERMADs. Proteins bound to the beads were fractionated by SDS-PAGE followed by western blotting using anti-ezrin or anti-moesin antibodies. CD44-CT was used as a positive control. (B) FRETeff values for cells coexpressing EYFP/Ezrin-ECFP (n=5), PWT-EYFP/Ezrin-ECFP (n=14), P{Delta}CT-EYFP/Ezrin-ECFP (n=7), PCTQN.N-EYFP/Ezrin-ECFP (n=7), PCTQN-EYFP/Ezrin-ECFP (n=6) and PCT.N-EYFP/Ezrin-ECFP (n=10). Note the significant reduction (**P<0.01) of FRETeff in PCT.N with respect to PWT and the absence of FRET in PCTQN.N, PCTQN and P{Delta}CT cell transfectants. (C-E) Confocal fluorescence images showing acceptor photobleaching FRET analysis in MDCK cell transfectants. Images of representative cells coexpressing Ezrin-ECFP with PWT-EYFP (C), P{Delta}CT-EYFP (D) or PCT.N-EYFP (E) are shown. ECFP and EYFP emission signals were collected before (left panels) and after (right panels) EYFP photobleaching in the boxed regions. An increased ECFP fluorescence signal after photobleaching indicates FRET. The FRETeff is represented in the bottom panel on a pseudocolor cell map with scale shown on the right.





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