Figure 5. Effects of vimentin head domain deletion on IbeA-induced NF-κB activation and interaction with β-tubulin.

(A) The cytoplasmic fractions of the GFP–VRT, GFP-VH and GFP transductants were extracted and immunoprecipitated (IP) using the mouse anti-GFP antibody as described in Materials and Methods. The GFP-IP complexes were subjected to Western blotting using the rabbit polyclonal antibodies against GFP, NF-κB (P65), and β-tubulin. Band a, GFP–VRT (72 kDa); band b, GFP-VH (37 kDa); band c, GFP (27 kDa); band d, NF-κB (P65), (65 kDa); and band e, β-tubulin, (50 kDa). (B) Immunofluorescence images of the GFP–VRT and GFP transductants incubated with or without the IbeA protein (0.1 µg/ml) for 2 h. The cells were double-stained with the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). Arrows indicate cells with NF-κB (P65) translocation to the nucleus, which was increased in the GFP transductants and reduced in GFP-VRT-transduced HBMECs upon stimulation with IbeA. Scale bar, 50 µm. (C) Western blot of the transduced HBMECs treated with the IbeA protein (0.1 µg/ml). ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, vimentin (VIM), GFP and PSF re-localization were examined in cytoplasmic fractions after 30 min of IbeA stimulation. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of IbeA incubation. β-actin in both fractions was detected as internal loading controls.