Analysis of endocytosed CD443MUT localization.
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(A) HUVEC were incubated with A488-labeled CD44HABD or CD443MUT (green) in the presence of CTxB-A555 (red) for 30 min. Nuclei were stained with Hoechst. Images show single confocal plane. Scale bars, 10 µm. (B) Colocalization analysis of CD44HABD (HABD) and CD443MUT (3MUT) with CTxB. Left, the fraction of CTxB-vesicles colocalizing with HABD (n = 39 cells) or 3MUT (n = 38 cells). Middle, the number of CTxB-vesicles per cell; right, the number of HABD- or 3MUT-containing vesicles per cell. (C–E) HUVEC were incubated with CD443MUT-A488 for 10 min after which CD443MUT-containing media was changed to 10% FBS HUVEC growth media and cells were further incubated for 20 min. Then cells were fixed and stained with anti-EEA1 or anti-CD63 antibodies. (C) Localization of 3MUT- and early endosomal marker EEA1-positive veicles after 10 min incubation in HUVEC. (D) Quantitation of EEA1-vesicles colocalizing with CD443MUT after 10 min incubation (n = 26 cells) and after 20 min chase (n = 40 cells; left). The number of EEA1- and 3MUT vesicles per cell (middle and left, respectively). (E) Localization of internalized 3MUT and late endosomal protein CD63-positive vesicles. Scale bars, 2 µm (C) and 5 µm (E).