Inhibition of syntaxin 6 function decreases the levels of VEGFR2 but not the levels of VEGFR1. (A,D) Uninfected (Control) and syntaxin 6-cyto or syntaxin 16-cyto expressing HUVECs (after 20 hours of infection) were stained with mAb against VEGFR2 (55B11), syntaxin 6 (in Control and syntaxin 6-cyto–treated cells) or syntaxin 16 (in syntaxin 16-cyto–treated cells). Samples were then fixed and observed under fluorescence microscope. (B) HUVECs were subjected to siRNA-mediated syntaxin 6 or syntaxin 16 knockdown, and immunostained for VEGFR2. Representative images showing staining for intracellular VEGFR2 in cells in which endogenous syntaxin 6 or syntaxin 16 was knocked-down over 90% after 72 hours of siRNA treatment. (C) Samples were fixed and observed as in panel A, but stained with goat pAb against VEGFR1 and antibodies against syntaxin 6 or syntaxin 16. (D) Quantification of intracellular VEGFR2 or VEGFR1 in syntaxin 6-cyto and syntaxin 16-cyto expressing cells, and in cells in which endogenous syntaxin was knocked down by siRNA treatment (as in panels A-C). Epifluorescence images were acquired and total cell-associated fluorescence was quantified by image analysis. Values represent relative change in the levels of VEGFR2 or VEGFR1 normalized to an arbitrary value of 100 for untreated controls. Percentage is expressed as mean (± SD) of n = 90 cells for each condition from 3 separate experiments; P ≤ .001. (E) Lysates were prepared from uninfected, syntaxin 6-cyto– or syntaxin16-cyto–infected HUVECs (after infection for various periods of time, as indicated) and samples were immunoblotted for VEGFR2 (55B11) and VEGFR1 (rabbit polyclonal). Relative level of endogenous syntaxin 6, syntaxin 16, or tubulin in cell lysate is shown. (F) VEGFR2 and VEGFR1 band density from panel E was quantified and results represent relative levels of VEGFR2 and VEGFR1 after normalization to an arbitrary value of 100 for 0 minutes after infection. Percentage is expressed as mean (± SD) for n = 3; P ≤ .005). Scale bar represents 5 μm.