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Fig. 8. The TGN targeting signal of VAMP4 plays a role in VAMP4-EGFP recycling. (A) The schematic illustration of expression constructs of VAMP4-EGFP and its mutants. Site-directed mutations were created in the context of VAMP4-EGFP. VAMP4-EGFP/LL-AA has the di-Leu motif (residues 25-26) replaced by two Ala residues. The first acidic cluster EDD (residues 27-29) was mutated into three Ala residues in VAMP4-EGFP/EDD-3A. VAMP4-EGFP/DEEED-5A has the distal acidic cluster (DEEED, residues 31–35) replaced by a stretch of five Ala residues. The two Phe residues at positions 36-37 were replaced by Ala residues in VAMP4-EGFP/FF-AA. NE, N-terminal extension; SNARE, SNARE domain; TM, transmembrane domain. These constructs were each transfected into NRK cells and pools of stable transfectants were selected and expanded for the experiments. (B) NRK cells expressing VAMP4-EGFP (a-c), VAMP4-EGFP/LL-AA (panels d-f), VAMP4-EGFP/EDD-3A (panels g-i), VAMP4-EGFP/DEEED-5A (panels j-l) and VAMP4-EGFP/FF-AA (panels m-o) were incubated at 4°C with rabbit anti-EGFP antibody in cold DMEM for 1 hour. After a brief wash, cells were then incubated in fresh DMEM at 37°C for 15 minutes. Panels a,d,g,j,m show the EGFP signal of these fusion proteins. Anti-EGFP antibody was detected by Cy3-conjugated goat anti-rabbit secondary antibody (b,e,h,k,n). The merged images are also shown (c,f,i,l,o). (C) Cells from B were washed twice in cold acidic washing buffer to strip off antibody remaining bound on the surface before being fixed for immunofluorescence microscopy. Panels a, d, g, j and m show the EGFP signal of these fusion proteins. Anti-EGFP antibody was detected by Cy3-conjugated goat anti-rabbit secondary antibody (panels b,e,h,k,n). The merged images are also shown (panels c,f,i,l,o). Bars, 10 µm.
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