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4, 150?mM NaCl, 1?mM EDTA, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS and proteinase inhibitor). The protein was removed from the beads by boiling for 5?min in 1X LSB (2% SDS, 10% glycerol, 6.25?mM Tris pH 6.8). Proteins were diglyceride separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk and then probed with 0.4?��g/ml goat polyclonal antibody against mouse BSG (R&D) overnight at 4?��C. The membranes were then washed and incubated with horseradish peroxidase-conjugated donkey anti-goat IgG antibody (abcam, San Francisco, CA) at 1:5000 dilution for 60?min at room temperature. The bound secondary antibody was detected using SuperSignal West Pico Chemiluminescent Substrate kit (Fisher Scientific). Membranes probed for immunoblotting analysis were stripped with Restore Western Blot Stripping Buffer (Fisher Scientific). After blocking in 1X Carbo-free blocking buffer (Vector Laboratories) for an hour, the membranes were incubated with 1??g/ml GSA II overnight at 4?��C. Membranes were then incubated in 0.2??g/ml Immunopure Avidin AZD0530 in vitro Hoseradish Peroxidase Conjugated (avidin-HRP) (Fisher Scientific) for 1?h. The bound avidin-HRP was detected using SuperSignal West Pico Chemiluminescent Substrate kit (Fisher Scientific). Methods for mouse spermatogenic cells (GC-2) and Sertoli cell (SF7) adhesion assays (Akama et al., 2002) were performed as previously described with the following modifications. SiRNAs corresponding to mouse Bsg (sense: CGUAGAUUCCCAUCAUACAtt; antisense: UGUAUGAUGGGAAUCUACGgg) were purchased from ambion (Life technologies). The siRNA was transfected into the GC-2 cells following the protocol of siLentFect? Lipid (Bio-Rad). Briefly, 3?��l of siLentFect Lipid transfection reagent was mixed with 10?nM of siRNA to form complexes and dispersed into 6-well cell culture plates at 37?��C for 48?h. Silencer select negative control #1 (Life technologies) was used as a negative control under the same conditions. The GC-2 cells were then labeled with [3H] Thymidine (2?��Ci/ml) at 37?��C for another 24?h. 4.2��105 [3H]-labeled GC-2 cells were added to the SF7 cells and incubated at 37?��C for 4?h. After washing unbound cells with PBS, radioactivity remaining on the SF7 cells was counted in a GDC941 scintillation counter. Results are represented as mean��S.E. Differences between groups were examined using Student's t-test. Statistical significance was set as P
