Cell lysis and immunoblotting For immunoprecipitation, 107 cells were lysed for 15 min at 4°C in a lysis Selleckchem AZD4547 buffer (50-mM Tris-HCl, pH 7.4, 150-mM NaCl, 5-mM EDTA, 10-mM NaF, 1-mM sodium orthovanadate, 1-mM phenylmethanesulfonyl fluoride, 1-μg/ml leupeptin, 1-μg/ml pepstatin, 1-μg/ml aprotinin and 1% Triton X-100). Total protein content in the lysates was determined using the Bio-Rad protein assay (Bio-Rad), and 150 μg of protein was incubated with protein A-agarose see more beads (Invitrogen) previously coupled with the corresponding antibody. The immune complexes were washed five times with cold washing buffer

(50-mM Tris-HCl, pH 7.4, 150-mM NaCl, 5-mM EDTA, 10-mM NaF, 1-mM sodium orthovanadate, 1-mM

phenylmethanesulfonyl fluoride, 1-μg/ml leupeptin, 1-μg/ml pepstatin, 1-μg/ml aprotinin and 0.1% Triton X-100) and resolved by SDS-PAGE (10% acylamide). To obtain total cell lysates, 107 cells were washed once with ice-cold phosphate-buffered saline (PBS) in a microfuge tube. Pellets were rapidly resuspended in 40 μL of lysis buffer, incubated for 15 min on ice and insoluble material was pelleted (15,000 × g for 15 min) at 4°C. Forty microliters of 2× Laemmli sample buffer (120-mM/L Tris, pH 6.8, 2-mM urea, 100-mM/L DTT, 10% glycerol and 0.001% bromophenol blue) were immediately added while vortexing, and the sample was boiled www.selleckchem.com/products/chir-99021-ct99021-hcl.html for 5 min. Fifty microliters of CHIR99021 each sample, along with molecular weight markers (Bio-Rad), were electrophoresed by vertical SDS-PAGE. The proteins were electroblotted onto nitrocellulose membranes, and the membranes were blocked overnight

in TBST buffer (10-mM Tris-HCl, pH 7.4, 100-mM NaCl and 0.5% Tween 20) containing 3% BSA. For protein immunodetection, the membranes were subjected to immunoblotting with 1 μg/ml of the appropriate antibody for 1.5 h at room temperature followed by HRP-conjugated anti-mouse or anti-rabbit IgG diluted to 1:6,000 (Zymed) for 30 min at room temperature. The membranes were then washed five times in TBST and the bands were visualized using the ECL system, according to the manufacturer’s instructions (Pierce). ELISA assay For ELISA assays, 5 × 104 U-937 and THP-1, as well as CALO and INBL, cells were plated in 48-well plates for 7 days. The cell culture supernatants were collected every 24 h and stored at -70°C until use, and ELISA detection was performed using 100 μL of each supernatant. In brief, plates were coated with 100 μL of the supernatants from the leukemic myelomonocytic and cervical cancer cells by incubating at 37°C for 1 h, washing three times with PBS-Tween (PBST) and blocking with 120 μL of PBST-3% BSA for 1 h at 37°C. Monoclonal antibodies (1:100 in PBST-3% BSA) were added for 1 h at 37°C. Anti-mouse IgG2a-HRP (1:4000 in PBST-3% BSA) was added for 1 h at 37°C. Plates were then washed and developed using 100 μL of ABTS system substrate (Zymed). The absorbance was measured at 405 nm.