Fig. 3.

HGF induces intracellular association of VEGF and VEGFR2.

(A and B) H441 cells were treated with vehicle or HGF (100 ng/ml) for 24 h, fixed and stained for immunofluorescence with anti-VEGF (green), anti-VEGFR2 (red) and DAPI (Nucleus, blue) (A), or anti-VEGF (green), anti-ER or Golgi marker (red), and DAPI (blue) (B). VEGF colocalization with VEGFR2, ER or Golgi was quantitated (B, right panel). Nuclear stain is DAPI (blue). Error bars indicate S.E.M., n = 3; **p < 0.01; *p < 0.05 for indicated comparisons.

(C) Cells were treated as above, and analyzed by proximity ligation assay (PLA) with quantification (right) with or without permeabilization. Red dots denote regions of signal amplification for VEGF:VEGFR2 proximity ligation. Nuclear stain is DAPI (blue). Error bars indicate S.E.M., n = 4; ***p < 0.005; *p < 0.05 for indicated comparisons.

(D) Cells were treated as in (C), permeabilized, and PLA was counterstained with either the ER marker (top) or Golgi marker (bottom). Green dots denote VEGF:VEGFR2 association. ER or Golgi stain is red and nuclear stain is blue. Colocalization between PLA signal and ER or Golgi staining is quantified (right). Error bars indicate S.E.M., n = 3; *p < 0.05, arrows denote colocalized signal.

(E) Cells were treated as above and PLA (green) was counterstained with indicated anti-Rab antibody (red), with nuclear stain (blue). Colocalization between PLA and Rab signals was quantified as in (D). Error bars indicate S.E.M., n = 3; **p < 0.01 for indicated comparison.