, 2004). ECV, treated or not with venom, were lysed in 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, Triton X-100 (1%, v/v), glycerol (10%, v/v), aprotinin (10 μg/μl), leupeptin (10 μg/μl), pepstatin (2 μg/μl), and 1 μM PMSF. Lysates (2 μg of protein/μl) were incubated overnight at 4 °C with rabbit polyclonal anti-FAK Ab (1:200). After that, protein A/G-agarose (20 μl/sample) was added, and samples were incubated at 4 °C in a rotatory shaker for 2 h (Nascimento-Silva et al., 2007). The contents

of click here FAK and actin associated to FAK were analyzed by immunoblotting as described below. The translocation of NF-kB to cell nucleus was analyzed by immunofluorecence microscopy and also by western blot detection of NF-kB p65-subunit in ECV nuclear extracts. For immunofluorescence studies, the ECV grown on glass coverslips and fixed with paraformaldehyde as described above, were blocked with 5% BSA/PBS for 30 min, and then incubated with rabbit polyclonal anti-p65 NF-κB Ab (1:50; Santa Cruz, sc-372; CA, USA) overnight at 4 °C. Subsequently, cells were washed three times with PBS and incubated with biotin-conjugated anti-mouse or anti-rabbit IgG (1:50) followed INK 128 research buy by incubation with Cy3-conjugated streptavidin (1:50)

for 1 h at room temperature. Coverslips were mounted on a slide using a solution of 20 mM N-propylgalate and 80% glycerol in PBS and examined under an Olympus BX40 microscope equipped for epifluorescence ( Nascimento-Silva et al., 2007). Nuclear extracts of ECV treated or not with L. obliqua venom were obtained as described. Briefly, cells were lysed in ice-cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF), and RVX-208 after a 15-min incubation on ice, Nonidet P-40 was added to a final concentration of 0.5% (v/v). Nuclei were collected by centrifugation (1810 × g; 5 min at 4 °C). The nuclear pellet was suspended

in ice-cold buffer C (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 μg/ml pepstatin, 1 μg/ml leupeptin, and 20% (v/v) glycerol) and incubated for 30 min. Nuclear proteins were collected in the supernatant after centrifugation (12,000 × g; 10 min at 4 °C), and the nuclear extracts were denatured in sample buffer (50 mM Tris–HCl, pH 6.8, 1% SDS, 5% 2-ME, 10% glycerol and 0.001% bromophenol blue) and heated in a boiling water bath for 3 min and assayed in SDS-PAGE ( Nascimento-Silva et al., 2007). Samples (30 μg total protein) were resolved by 12% SDS-PAGE and proteins were transferred to PVDF membranes for western blot analysis (Nascimento-Silva et al., 2007). Molecular weight markers were run in parallel to estimate molecular weights. Membranes were blocked with Tween-TBS (20 mM Tris–HCl, pH 7.5; 500 mM NaCl; 0.