Localization of tTG on the surface of THP-1 macrophages adherent on Fn.
THP-1 monocytic cells were induced with 150 ng/mL TPA and plated on Fn-coated coverslips for 72 hours. Live nonpermeabilized cells were double-stained for β1-integrins and tTG (A), β3-integrins and tTG (B), or FcγRI receptor and tTG using polyclonal anti-tTG antibody (C). Note an extensive colocalization of tTG with β1- and β3-integrins on the surface of THP-1 macrophages and their specific accumulation in podosomes (arrows, arrowheads). Alternatively, live nonpermeabilized cells were stained for surface tTG with polyclonal antibody, fixed, permeabilized, and then costained for F-actin (D) or vinculin (E). The mAb 4G3 also revealed localization of tTG in podosomes of THP-1 macrophages (F), whereas incubation without primary and with secondary antibody only (fluorescein isothiocyanate–labeled anti–rabbit IgG) resulted in negative background staining and no detectable staining of podosomes (G). Arrows point to ringlike and arrowheads mark round-shaped podosomes. Asterisk marks a podosome containing β3-integrins, but little or no tTG. Bar indicates 25 μm.
