Sulfo-SBED-labeled DNT (SBED-DNT), which had a similar distribution to the native toxin (Fig. 1A-d), transferred biotin to at least three distinct cellular components in NP-40 insoluble fraction detected by Western blotting (Fig. 1C). Only the component with the highest molecular weight could be isolated by anion-exchange chromatography (Fig. 1D and 1E), and identified as mouse FN by mass spectrometry. FN is a major component organizing the ECM. We examined if the toxin

colocalizes with the FN network by staining FN or other ECM components, such as collagen type I and laminin. DNT was found to be well colocalized with the FN network and partly colocalized p53 activator with the collagen type I, but not colocalized with laminin (Fig. 2). Figure 1 DNT is associated with the fibrillar structure on MC3T3-E1 cells. (A) The cells were treated with DNT (a and b), Selleck Navitoclax 5-FAM-DNT (c), or SBED-DNT (d) as mentioned in Methods. The cells were stained without wash as follows. DNT was detected with a combination of anti-DNT polyclonal antibody and Alexa 488-conjugated secondary selleck products antibody (b). The DNT-treated cells were stained with only the secondary antibody for the control (a). 5-FAM-DNT was visualized with direct fluorescence microscopy (c). SBED-DNT was detected with Alexa 488-conjugated streptavidin

(d). Note that the association of DNT with the fibrillar structure was observed independently of the detection method. Bar, 5 μm. (B) MC3T3-E1 cells were incubated with DNT at different pH and stained with anti-DNT polyclonal antibody. The cells were washed once (lower panels) or not washed (upper panels) before fixation. Bar, 5 μm. (C) Cellular components cross-linked by SBED-DNT. MC3T3-E1 cells were incubated with (lane 2) or without (lane 1) SBED-DNT. After the cross-linking procedure, the insoluble fraction was prepared as described in Methods and subjected to SDS-PAGE with a 6% acrylamide gel containing 6 M urea under

reducing conditions. Cellular components labeled by biotin through SBED were detected by Western blotting with HRP-conjugated streptavidin. Arrows indicate cellular components cross-linked Org 27569 with SBED-DNT. (D) Mini Q column chromatographic profile of the insoluble fraction of MC3T3-E1 cells treated and cross-linked with SBED-DNT. The cellular component with the higher molecular weight was eluted in fractions 6 to 8 (bold bar). (E) SDS-PAGE of fraction 7. The cellular component with the higher molecular weight is indicated with an asterisk. Figure 2 Colocalization of DNT with the ECM components. MC3T3-E1 cells incubated with DNT were stained with anti-DNT monoclonal antibody or polyclonal antibody against FN, collagen type I or laminin. Bars, 5 μm. Besides MC3T3-E1 cells, which are sensitive to DNT, DNT-insensitive Balb3T3 cells also showed the colocalization of DNT with the FN network (Fig. 3).