Proteins were transferred on to nitrocellulose membranes (catalogue no

Proteins were transferred on to nitrocellulose membranes (catalogue no. 0.01?vol. of culture supernatant of M-CSF-producing cells and 100?ng of GST-RANKL (glutathione S-transferase-RANK ligand) [11]. In osteoclastogenesis assays involving the use of the chimaeric receptor, human Fas-activating antibody (Upstate Biotechnology, Lake Placid, NY, U.S.A.) was added at concentrations as indicated in individual assays. Osteoclasts were stained for TRAP (tartrate-resistant acid phosphatase) activity using a commercial kit (387-A; Sigma). Bone resorption assays were performed by generating osteoclasts on whale dentin slices from infected or uninfected BMMs as described above. Dentin slices were harvested 4C5?days after the osteoclasts were formed. Cells were removed from the dentin slices with 0.25?M ammonium hydroxide through mechanical agitation. Dentin slices were then subjected to scanning electron microscopy as described in [12]. Flow cytometric analysis Infected BMMs (up to 1106?cells) were suspended in 200?l of PBS/azide. Cells were then blocked with 1?g of 2.4G2 antibody [13] for 30?min on ice. Under dim light, 20?l of anti-human Fas antibody conjugated with phycoerythrin (sc-21730PE; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) or control IgG was added to the cell suspension and Thiomyristoyl cells were incubated on ice for 30?min. Cells were washed twice with 1?ml cold PBS/azide and resuspended in 300?l cold PBS/azide. Cold 0.5% of paraformaldehyde solution (200?l) was added to fix the cells. Flow cytometric analysis Thiomyristoyl was performed using a Becton-Dickinson FACSan (Becton-Dickinson Immunocytometry Systems, Mountain View, CA, U.S.A.). Western-blot analysis Infected or uninfected BMMs were cultured in serum-free -MEM in the absence of M-CSF for 16? h before treatment with RANKL or Fas-activating antibody. Cells were washed twice with ice-cold PBS and then lysed in a buffer containing 20?mM Tris (pH?7.5), 150?mM NaCl, 1?mM EDTA, 1?mM EGTA, 1% Triton X-100, 2.5?mM Thiomyristoyl sodium pyrophosphate, 1?mM -glycerophosphate, 1?mM Na3VO4, 1?mM NaF, and 1protease inhibitor cocktail 1 (P-2850; Sigma) and 1protease inhibitor cocktail 2 (P-5726; Sigma). Western-blot analysis was performed largely as described in [14]. Cell lysates (40?g) were boiled in the presence of SDS sample buffer [0.5?M Tris/HCl, pH?6.8, 10% (w/v) SDS, 10% (v/v) glycerol and 0.05% (w/v) Bromophenol Blue] for 5?min and loaded on to SDS/10% polyacrylamide gel for electrophoresis. Proteins were transferred on to nitrocellulose membranes (catalogue no. 162-0147) from Bio-Rad (Hercules, CA, U.S.A.) using a semi-dry blotter (Bio-Rad). Membranes were blocked in blocking solution [5% (v/v) non-fat dried milk in TBS (Tris-buffered saline) containing 0.1% Tween 20] for 1?h to prevent nonspecific binding and Thiomyristoyl then washed three times with TBS-T (TBS containing 0.1% Tween 20). Membranes were incubated with primary antibodies in TBS-T containing 5% bovine albumin (Sigma; catalogue no. A-7030) overnight at 4?C. Next day, membranes were washed three times with TBS-T and incubated with secondary antibody in TBS-T containing 5% nonfat dried milk for 1?h. Membranes were washed extensively and chemiluminescence detection assay was performed using SuperSignal West Dura kit from Pierce (Rockford, IL, U.S.A.). RESULTS Construction of a chimaeric FLJ25987 receptor containing the human Fas external domain for studying signalling by TNFR family members Although many members of the TNFR family are believed to be activated by ligand-induced trimerization [1], recent results indicated that some TNFR family members, including Fas, are self-assembled as trimers before ligand binding and the.