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Fig. 1. CD317 is internalised through a clathrin-dependant pathway. (A) H4IIE cells transiently expressing CD317-GFP were imaged and the highlighted area was photo-bleached and allowed to recover for 17 minutes. Images were taken every minute, Cells were incubated in the presence of 200 µg/ml cycloheximide for 1 hour prior to imaging (and in the continued presence of cycloheximide during imaging). Bar, 10 µm. (B) Transiently transfected H4IIE cells expressing dominant-negative AP180-C-myc were subjected to uptake of transferrin (Tfr), cholera-toxin-B subunit (CT-B) or CD317 antibody uptake for 15 minutes: cells were then acid washed to remove any non-internalised material. Asterisks denote transfected cells. Bars, 10 µm. (C) Immunoblot of fractions obtained during the isolation of purified clathrin-coated vesicles (CCVs) from rat brain, which had been separated by SDS-PAGE (12% gel), using anti-CD317 antibody (upper panel), or anti-GSK3 antibody (lower panel). Lane 1, purified CCVs; lane 2, peak 1 from the purification of CCVs (containing vesicles depleted in CCVs); lane 3, cytosol; lane 4, post-nuclear fraction; lane 5, total rat brain lysate. The same blot was initially probed with anti-GSK3 antibody, stripped and re-probed with anti-CD317 antibody. The fractions are the same as those previously described and characterised (Korolchuk and Banting, 2002). (D) HeLA cells transiently transfected with a construct encoding wild-type CD317. After 24-hour-expression, cells were subjected to antibody uptake (using a CD317 antibody) and Tfr uptake for 2 minutes, or just antibody uptake for 6 minutes. Images show colocalisation between EEA1 or Tfr and CD317 as indicated. Indicated areas have been enlarged. Colocalisation between EEA1 and CD317 is 64.1% (n=310) and between Tfr and CD317 40% (n=233). Bars, 10 µm.
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