Fig. 9. BAD-LAMP is targeted to early endosomes and recycles in HeLa cells. HeLa cells transfected with FLAG-BAD-LAMP were submitted to immunofluorescent staining and confocal microscopy visualization. (A) FLAG-tagged BAD-LAMP (anti-flag antibody, red) was found at the cell surface and in internalized transferrin-FITC-containing endosomes. Cytoplasmic tail tyrosine 276 mutant (Tyr276-Ala) was found accumulating at the surface of transfected cells (anti-flag antibody, red) with little intracellular distribution (transferrin-FITC, green). (B) Transfected FLAG-BAD-LAMP is not detected in LAMP1- (blue) and DC-LAMP (green)-positive late endosomes and lysososomes. (C) Kinetics of FLAG antibody uptake after cold binding on the surface of transfected HeLa cells. Only transfected cells accumulate the antibody (red) on their surface, which upon warming reaches rapidly sorting (5 minutes) and recycling (15 minutes) endosomes containing transferrin-FITC (green). No co-localization with LAMP1 (white, 45 minutes) could be observed, suggesting that BAD-LAMP and associated antibodies do not access the late endocytic pathway. (D) Co-expression of dynamin dominant negative mutant A44K (right panel, green) in FLAG-BAD-LAMP-transfected HeLa cells prevents the internalization of associated flag antibodies (right panel, red), whereas expression of wild-type dynamin has no effect (green, left panel). (E) Co-transfection of HeLa cells with FLAG-BAD-LAMP (anti-BAD-LAMP, red) and pSuper control plasmid (left) has no effect on the internalization of associated flag antibodies (green). Conversely RNAi inhibition of the clathrin adaptor AP2 blocks flag antibodies uptake (green). Bars, 20 µm.