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 buy ICG-001 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), 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 PD0325901 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 Ibrutinib order 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).