Abl and Arg are activated in EPEC pedestals. (a–h) Images of 3T3 cells exposed to EPEC and stained with DAPI (a), FITC-phalloidin (b), and α-pY412 pAb (c). In the merged images (d and h), EPEC are pseudocolored blue, actin green, and pY412 red. Note the presence of α-pY412 staining in the pedestals. Images in e–h are enlargements of pedestals found in a–d, as indicated by the white box in b. (i–l) Images of Abl-/-/Arg-/- cells stained with DAPI (i), FITC-phalloidin (j), α-HA (k), and α-pY412 (l). Note that although pedestals form in Abl-/-/Arg-/- cells, no pY412 staining was evident. (m–p) Images of Abl-/-/Arg-/- cells expressing HA-Abl and stained with DAPI (m), α-HA (n), α-pY412 (o), and FITC-phalloidin (p). (q–t) Images of Abl-/-/Arg-/- cells expressing Arg-YFP and stained with DAPI (q) and α-pY412 pAb(s). The YFP channel is shown in r. Note that transfected Abl or Arg colocalize with PY412 staining. Together, these data suggest that α-pY412 pAb recognizes phosphorylated Abl or Arg but not other pedestal proteins and that both Abl and Arg are activated in pedestals. Bars, 5 μm.