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 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 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.