The insoluble fraction was sonicated in D-PBS (-) containing 5 μg/ml of DNase I and 8 M urea. After centrifugation, the supernatant was injected into a
Mini Q column (0.32 × 3 cm, GE Healthcare), and eluted with a gradient of 0-1 M NaCl in 20 mM Tris-HCl (pH 8.5), containing 8 M urea, this website using the SMART system (GE Healthcare). Screening for components intermediating the association between DNT and the FN network FN-null cells or MC3T3-E1 cells were cultured in FCS-free DMEM or α-MEM for 72 h. The supernatant of the culture was dialyzed against 20 mM Tris-HCl, pH 8.5 containing 0.5 M NaCl, and subjected to anion-exchange chromatography with a HiTrap Q column (0.7 × 2.5 cm, GE Healthcare). The materials absorbed to the column were eluted in 1-ml fractions with a linear gradient of 0.5-1 M NaCl, and each fraction was tested for the ability to recruit DNT to the fibrillar structure on MRC-5 cells using immunofluorescence microscopy. The positive fractions were collected, appropriately diluted, and mixed with 5% CHAPS and 10 M urea to make a solution of 20 mM Tris-HCl, pH 8.5, containing 50 mM NaCl, 0.5% CHAPS and 6 M urea. The sample was subjected to Mono Selleck SAHA HDAC Q anion-exchange chromatography, and eluted with a linear gradient of 0.05-1 M NaCl. The eluted fractions were examined again for the ability to recruit DNT to the fibrillar structure on MRC-5 cells. Proteins contained in the positive fraction were identified
by mass spectrometry as mentioned below. DNT diffusion assay FN-null cells were seeded in wells of a 24-well plate at 25,000 cells/cm2 and grown overnight. The next day, the cells were washed well with Cellgro-Aim V and incubated overnight in the same medium with PRKACG or without 10 μg/ml of human FN. The culture medium was replaced with a fresh batch containing 2.5 μg/ml of DNT and the cells were incubated for 15 min at 37°C. After three
washes with FCS-free DMEM, the cells were further incubated in the fresh medium. The culture supernatant was taken at the indicated time point, and an aliquot was selleckchem applied to MC3T3-E1 cells without dilution. After incubation at 37°C overnight, the cells were examined for actin stress fibers as described previously . Another aliquot of the culture supernatant was examined for DNT by sandwich-ELISA, performed with a 96-well plate coated with anti-DNT polyclonal antibody. After blocking with 0.2% BSA at 4°C overnight, each sample was added to the plate in triplicate and incubated for 2 h at 37°C. The plate was treated with biotin-labeled anti-DNT antibody, followed by HRP-conjugated streptavidin for 1 h at 37°C. BM Blue POD substrate (Roche) was used as an HRP substrate and the reaction was stopped by 1 M H2SO4. The wells were washed four times with D-PBS (-) containing 0.05% Tween-20 between each step. The concentration of DNT was estimated from a standard curve made with a DNT preparation. Other methods Protein concentrations were determined using BCA Protein Assay Reagents (Thermo Scientific).