Inhibition of syntaxin 6 function targets VEGFR2 to the lysosomes for degradation. (A,B) Uninfected (Control) and syntaxin 6-cyto– or syntaxin 16-cyto–infected HUVECs (20 hours of infection) were metabolically labeled with ³⁵S-methionine–³⁵S-cysteine for 20 minutes. After the indicated chase period in an excess of unlabeled methionine-cysteine, the cells were lysed and total cell-associated precipitated. Radioactive counts were normalized before immunoprecipitation with VEGFR2 antibody (55B11). Autoradiographic signals from films were measured using Image J software Version 1.38x (NIH) and are represented as a percentage of the time 0 values for uninfected controls. Values are mean (± SD) for n = 3; P ≤ .05. (C,E) After 18 hours of infection, uninfected (Control) and syntaxin 6-cyto– or syntaxin 16-cyto–treated HUVECs were incubated for an additional 6 hours in complete medium (with 0.05% dimethyl sulfoxide [control], 100μM CHQ, 100nM Baf, or 5μM Lac). Samples were then costained with antibodies against VEGFR2, EEA1 (data not shown), or Lamp2 and observed by fluorescence microscopy. Fluorescence images were used to quantitate total cell-associated VEGFR2 (D) or the colocalization of VEGFR2 with EEA1 or Lamp2 (E). Values in panels D and E represent mean (± SD) for n = 80 cells for each condition from 5 separate experiments; P ≤ .05. Scale bar represents 5 μm.