Image las4010

HT-29, PANC-1, MDA-MB-231, and MCF-7 cells were seeded in 6-well plates (1.5 × 10⁶ cells per well), previously coated with 10 μg/ml plasma fibronectin, and maintained overnight in a CO₂ incubator. Cells were lysed according to the antibody supplier's instructions (Abcam). Total proteins (20 μl/lane) were separated with a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (GE Healthcare). The membrane was saturated with 5% w/v nonfat dry milk in PBS containing 0.1% Tween20 for 1 h at room temperature and then incubated with specific antibodies [rabbit polyclonal to sulfatase 1/SULF-1 antibody (1 μg/ml, Abcam), and mouse anti-GAPDH monoclonal antibody (1 μg/ml, Invitrogen)]. After washing, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000, Cell Signaling) in the case of anti-sulfatase 1/SULF-1 antibody and with horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG) (1:10,000, ThermoFisher). Signals were detected using Image LAS4010 (GE Healthcare). Densitometry analysis was carried out using ImageJ software. The value 100% corresponds to average GAPDH protein expression for the four cell lines. The experiment was performed three times. P values were calculated using a parametric, unpaired Student t-test, and GraphPad Prism 5.0 software.

RAW264.7 cells were seeded in 6-well plates (3 × 10⁶ cells/well) and cultured overnight in a CO₂ incubator. Cells were stimulated with 25 ng/ml LPS from K. pneumoniae in the presence of 10 μm SET-M33 in DMEM for 6 h at 37 °C. After incubation, cells were washed twice with PBS and detached with ice-cold PBS. They were then centrifuged, suspended in lysis buffer (20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% cholic acid, and protease inhibitor mixture) and incubated for 30 min on ice. Products of cell lysis were centrifuged at 12,000 rpm for 10 min at 4 °C, and the concentration of proteins in supernatant (cytoplasmic extract) was determined by Bradford assay (Bio-Rad). 20 μg of total proteins was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked in PBS-5% BSA for 1 h at room temperature and then incubated overnight at 4 °C with antibodies specific for NF-κB p65 (Cell Signaling Technology) and for β-actin (Sigma-Aldrich) diluted in PBS-5% BSA-0.1% Tween 20. After washing with PBS-0.05% Tween 20, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma-Aldrich). Signals were detected using Image LAS4010 (GE Healthcare).