Effect of TNF-α/IFN-γ on the subcellular localization of JAM-A. (A) HUVECs were incubated for 16 hours in the absence or presence of TNF-α/IFN-γ and GW254023X. Triton X-100–soluble and –insoluble JAM-A was analyzed by Western blotting using an anti–hJAM-A monoclonal antibody. Detection of β-actin in the lysates confirmed equal loading. (B) Confluent HUVECs were incubated for 16 hours with TNF-α/IFN-γ in the absence or presence of GW254023X (GW) or vehicle control (DMSO). Samples were fixed and stained with anti–hJAM-A monoclonal antibody, followed by Alexa Fluor 488–conjugated goat anti–mouse IgG secondary antibody (green) and counterstaining of nuclei (blue).