Materials and Methods round cell cultures Monoclonal antibody Body against ERK2 directed Perk, CDK2, and caspase 3 were from Santa Cruz Biotechnology. The polyclonal antibody Body against SAPK / JNK and pSAPK / JNK were obtained from Cell Signaling. EGF, EGFR-selective inhibitor AG 1478, selective MEK inhibitor PD 98059, selective SAPK / JNK inhibitor SP 600125, hydroxyurea, YN968D1 EGFR inhibitor and the monoclonal antibody Body were used against b-actin in the study obtained from Sigma. Glycogen synthase kinase-3 serine 9 and polyclonal antibody ß Body against versican V1 were obtained from Abcam. Horseradish peroxidase-conjugated goat anti-mouse IgG conjugated with horseradish peroxidase and goat anti-rabbit IgG were obtained from Bio Rad. Immunoblotting was performed using the ECL Western blotting detection kit.
Cell proliferation reagent WST 1 was obtained from Roche Applied Science. Mouse mammary tumor cell lines 67NR, 66c14, 4Q07, 4T1 ATPase kinase and human breast cancer cell line MDA MB 231 were grown in DMEM cell line and human breast cancer MT 1, MCF-7, MDA MB 468 in RPMI 1640 supplemented with 10% f calf serum fetal K, penicillin and streptomycin was erg complements and grown at 37uC in a humidified atmosphere of 5% re CO 2. In select experiments, cell suspensions with EGF, the EGFR inhibitor AG-1478, selective MEK inhibitor, PD 98059, selective and SAPK / JNK inhibitor SP 600 125 cultivated. The construction and pcDNA1 pcDNA1 G3 G3 fragment lacking the GEF as a building Building designs were created by us.
Mouse mammary tumor cell lines 66c14 4Q07, the 4T1 cell line and human breast cancer MT 1, MDA-MB 231, MCF-7 and MDA-MB 468 cells were transfected with pcDNA1 constructs vecor and G3. The cells were transfected fa If the transition time with 66c14 G3 construction G3DEGF construction or vector control On. A sequence of the first plan, which was shown to be effective in the secretion product was con U both built by us before. Zelllebensf Higkeitstests G3-and vector-transfected cells were cultured in 66c14 10% FBS / DMEM in Bo Their culture and at 37uC for 12 hours. After cell attachment, we have the serum-free medium or DMEM 10% FBS / DMEM with various concentrations of chemotherapeutic compounds. The cells were t Resembled harvested and the number of cells was analyzed by Coulter counter-Z. Cell survival assays were performed with colorimetric proliferation assay.
Versican G3-and vector-transfected control breast cancer cells were seeded in 10% FBS / DMEM at 96 bo Their culture for 12 hours. After cell attachment, we have the serum-free medium or DMEM 10% FBS / DMEM containing various concentrations of chemotherapeutic agents, and the cells cultured with 10 ml of the reagent WST 1 for 4 hours. The absorbance of the sample against a contr The white S background was measured with a microplate Leseger t. Western blot of the protein samples for analysis were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gel to the separation of gel with 7 10% acrylamide. The separated proteins Were blotted onto a nitrocellulose membrane in a buffer containing 20% methanol 16Tris/glycine at 60 V for 2 h in a cold room. The membrane was not in TBST with 5% skim milk powder dry blocked for 1 hour at room temperature and then incubated with primary Ren Antique Rpern against 4UC night. The membranes were washed with TBST and then with appropriate horseradish peroxidase conjugated secondary Ren Antique Rpern