e serially diluted and plated on 5% horse blood agar plates and then incubated anaerobically at 37 C for 10 days. Colony forming units of invasive P. gingivalis in cells were then enumerated. Silencing of Rab5 gene Ca9 22 cells were transfected with 100 pmol siRNA spe cific for Rab5 or control siRNA using Lipofectamine 2000 reagent, as described by the manufacturer. Then, e pres sion of Rab5 in the cells was e amined by Western blotting using a monoclonal antibody to Rab5. Ne t, Rab5 siRNA transfected Ca9 22 cells were incubated with P. gingivalis ATCC 33277 for 1 h. Viable P. gingivalis in the cells was determined as described above. Immunostaining Treated Ca9 22 cells were fi ed with 4% formaldehyde for 10 min. Nonspecific binding of antibodies was blocked by incubation with 5% sheep serum in 10 mM Tris pH 7.
6, 150 mM NaCl, and 0. 05% Tween20 for 1 h, and then the cells were incubated overnight at 4 C with a primary antibody in TBS T. After washing with buffer A 6 times, the cells were treated with a secondary antibody in buffer A for 1 h. Cells were then observed by a confocal laser scanning microscope. Cilengitide Some Ca9 22 cells were transfected with vectors containing genes of GFP alone, GFP Rab5, and GFP Rab5. To clarify whether P. gingi valis cells are in the epithelial cells, a z series with 0. 5 um intervals was scanned and images of the z and y z planes were reconstructed with the orthogonal section tool. Western blotting TNF treated and non treated Ca9 22 cells and THP 1 cells were lysed in SDS PAGE sample buffer, separated by SDS PAGE, and transferred onto Immobilon P Transfer Membranes.
The membranes were blocked with PVDF Blocking Reagent for Can Get Signal in TBS T for 1 h at room temperature and then incubated with antibodies to TNFRI, TNFRII, Rab5 and ICAM 1 overnight at 4 C. After washing 3 times with TBS T, the membranes were incubated with horseradish pero idase conjugated anti rabbit or mouse IgG antibodies in Can Get Signal Immunoreaction Enhancer Solution. The membranes were washed 3 times with TBS T and then immunoreactive bands were visualized using ECL Western Blotting detection reagents or Immuno Star LD. The membranes were stripped and probed with anti B actin antibodies as a loading control. GST R5BD pull down assay The GST R5BD pull down assay was based on the method described by Liu et al.
Ca9 22 cells were transfected with GFP Rab5 using Lipofectamine 2000 reagent, as described by the manufacturer. The trans fectants were pretreated with MAP kinase inhibitors, in cluding a p38 inhibitor, JNK inhibitor, and ERK inhibitor, or with an NF ��B inhibitor at 37 C for 1 h followed by stimulating with 10 ng ml TNF for 3 h. Thereafter, cell e tracts were prepared in lysis buffer con taining 25 mM HEPES pH 7. 4, 100 mM NaCl, 5 mM MgCl2, 0. 1% Nonidet P 40, 2% glycerol, 1 mM dithio threitol, and protease inhibitors. The cell lysates were centrifuged at 13,000 g for 10 min at 4 C, and then the supernatants were incubated with 20 ul of G