The canine lines and human line SJSA were main tained in RPMI 1640 supplemented

After centrifugation at 14 000 × g for 5 min at 4 C, the supernatants were stored at 80 C. For pull down assays, 100 200 ug of nuclear protein extracts were incubated for 30 min at 4 C in binding buffer AP24534 溶解度 containing salmon sperm DNA and 1 ug of the biotinylated hairpin decoy ODN or the mutated decoy ODN. The complexes were captured by incubation with 50 ul of avidin Sepharose beads for 2 h at 4 C. For in cell decoy ODN pull down assays, the cells were first transfected with STAT3 decoy ODN or its mutated equivalent, as described under oligonucleotide transfection, and then processed as above by cell lysis and recovery on avidin Sepharose beads. After extensive washing with binding buffer, complexes were separated on SDS polya crylamide gel, and subjected to immunoblotting using an anti STAT3 antibody.

Results were analyzed by chemiluminescence and autoradiography. Antibody co immunoprecipitation For antibody pull down assays, 20 million cells were lyzed and resuspended in lysis buffer AT7519 臨床試験 at 4 C for 5 min. The lysates were centrifuged at 14 000 × g for 5 min at 4 C, and the supernatants containing the cyto plasmic proteins were either used immediately or stored at 80 C. For the immunoprecipitations, 200 to 400 ug protein was supplemented with albumin saturated pro tein G agarose, after centrifugation, the pellet was discarded and the supernatant conserved. Antibody was added and incubation continued overnight at 4 C. Samples were then supplemented with albumin saturated protein G agarose and incubated for 1 h 30 m. The agarose beads were washed three times with TBS and once with TBS T, and resuspended in SDS sample buffer.

Alisertib Aurora キナーゼ 阻害剤 Gel separation and western blotting were performed as described above. Results Survival of SW 480 colon carcinoma cells requires activated STAT3 STAT3 is activated in colon carcinoma and in the colon carcinoma cell line SW 480. In SW 480 cells transfected with specific STAT3 siRNA, the expression of STAT3 was strongly reduced and the number of annexin V positive cells was significantly increased in comparison to cells treated with control siRNA. STAT3 tyrosine 705 phosphorylation was detected as previously reported. Pull downs with biotinylated ODN were performed, fol lowed by western blotting with anti STAT3 antibody. This method is analogous to gel retardation assays, and revealed that STAT3 is activated.

ODN bound activated STAT3 was detected in nuclear extracts from untreated and IL 6 treated cells, cyto plasmic STAT3 only weakly bound the biotinylated ODN. Treatment of cells with the STAT3 inhibitor Stattic, known to inhibit STAT3 phosphorylation and dimerization, inhibited the nuclear translocation of STAT3 in SW 480 cells, as pre viously shown in other cell lines, this correlated with induction of cell death. Cytoplasmic sequestration of STAT3 and phospho STAT3 by STAT3 decoy ODN The subcellular distribution of STAT3 decoy ODN in SW 480 cells was shown by fluorescence microscopy to be essentially cytoplasmic. Immunofluorescence microscopy analyzis of the subcellular localization of phospho STAT3 in untreated SW480 cells showed that it was essentially nuclear, but following STAT3 decoy ODN treatment it became mostly cytoplasmic, this was not observed when using mutated STAT3 decoy ODN.