HM1.24 is internalized from lipid rafts by clathrin-mediated endocytosis through interaction with alpha-adaptin - PubMed (original) (raw)

Clathrin-dependent endocytosis of HM1.24. A, HeLa cells were transiently transfected with either an expression plasmid encoding GFP-Dyn2-WT (wild type; A, B, E, F, I, J, M, and N) or GFP-Dyn2-K44A (dominant-negative form; C, D, G, H, K, L, O, and P). After 24 h, cells preincubated with Cy3-Tfn (A–D), Rh-EGF (E–H), HM1.24 antibody (I–L), or anti-CD59 antibody (M–P) for 30 min at 4 °C were further incubated at 37 °C for 10 min and fixed. Non-internalized ligand was removed by washing at pH 3.0, 4 °C before fixation. Cells incubated with HM1.24 antibody and anti-CD59 antibody were permeabilized, and stained with Cy3-conjugated secondary antibody. Cells were then visualized by confocal microscopy. Bars, 10 μm. B and C, HeLa cells were treated without (A–C and J–L) or with 10 m

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MβCD (D and E, and M–O) or 25 μg/ml nystatin (G–I and P–R) at 37 °C for 30 min. Subsequently, cells preincubated with HM1.24 antibody (A, D, G, J, M, and P) and Cy3-Tfn (B, E, and H) or CD59 antibody (K, N, and Q) at 4 °C for 30 min were incubated at 37 °C for 5 min, acid washed at 4 °C, fixed, and processed for immunostaining. Right columns show merged images for double staining of HM1.24 antibody (green in A, D, and G; red in J, M, and P) and Cy3-Tfn (red) or CD59 antibody (green). Bars, 10 μm.