P8340, purchased by Sigma Aldrich)], and cell lysate was centrifuged at 14,000 �� g at 4��C for 20 min. The supernatant was harvested and analyzed for protein content using protein assay dye. Protein was denatured in sample buffer, then separated on SDS-PAGE, and transferred to polyvinylidene www.selleckchem.com/products/nutlin-3a.html difluoride membranes using a semidry trans-blot system. The blots were blocked for 1 h at room temperature with Tris-Buffered saline (TBS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 5% non-fat milk. The blots were washed three times with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.02% Tween 20) and incubated with the indicated antibody at 4��C overnight. Next day, the blots were incubated for 1 h at room temperature with secondary antibody (1:5000 dilutions), and detected by ECL detection reagent.

To ensure that equal amounts of sample protein were applied for electrophoresis, ��-actin was used as an internal control. Gene silencing The siRNA duplexes specific for human c-Abl (cat. no. L-003100-00) or p73 (cat. no. L-003331-00) were obtained from Dharmacon RNA Technologies. The siRNA for each group contained four RNA sequences in a Smart Pool selected from the NCBI RefSeq Database by a proprietary algorithm. The control non-targeting pool is a pool of four functional non-targeting siRNAs with guanine cytosine contents comparable to that of the functional siRNA but lacking specificity for known gene targets. To achieve gene silencing, we transfected cells with the indicated siRNA for 24 h followed by drug treatment; then the gene silencing effects were evaluated by Western blot analysis.

Immunoprecipitation For immunoprecipitation experiments, cells were washed with ice-cold PBS once and then lysed in 1 ml RIPA lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 2 mM NaF, 2 mM ��-glycerolphosphate, 2 mM Na3VO4, 1 mM PMSF, and protease inhibitor cocktails) and centrifuged at 10,000 rpm, 4��C for 5 min. The supernatant was collected and was pre-cleaned with 0.5 ��g normal IgG and 10 ��l protein A-agarose beads at 4��C for 30 min for each sample. After centrifugation, supernatant was incubated with specific antibody at 4��C overnight, and then 10 ��l protein A-agarose beads were added and rocked for another 1 h. The immunocomplexes were washed two times with cold RIPA buffer containing 150 mM NaCl, two times with RIPA buffer containing 300 mM NaCl and finally RIPA buffer containing 150 mM NaCl again.

SDS gel-loading buffer was added to the precipitated complexes and heat the samples at 95��C for 5 min. After spinning down the samples and loading the supernatants onto the SDS-PAGE, immunoblotting analysis AV-951 was performed as described above. In vitro c-Abl kinase assay To evaluate kinase activity of c-Abl, HCT116 cells were lysed in Tris-buffered saline-0.