The cell suspension was ana lyzed on a flow cytometer within 48 hours and ModFi

Lysates were spined down by centri fugation at 14,000 rpm for 30 min at 4 C, and lysate protein concentrations were determined using purchase JNJ-7706621 a Bio Rad protein assay, Electrophoresis and immunoblotting were per formed as described previously, with hybridization signals detected by chemiluminescence and captured using a FUJI LAS1000 plus chemiluminescence ima ging system. Immunoprecipitation Ovarian cancer cell lysates were prepared after serum starved for 2 h or treatment with 1 17 AAG in serum free medium for 6 h. One mg of protein lysate was precleared for 30 min using 30 ul of protein G or protein A beads at 4 C. Two ug of EGFR, ERBB2, ERBB4, MET, or AXL antibody was added to the super natants and rocked for 2 4 h at 4 C.<br><br> Then 25 uL of sepharose protein G or protein A beads were added and rocked overnight at 4 C, then centrifuged at 14,000 rpm for 2 min at 4 C, after which the sepharose beads were washed 3 times with 750 uL of IP buffer and once with 750 uL 10 オーダー LDN193189 mM Tris Cl buffer, Loading buffer was added to the beads and boiled for 5 min at 95 C. Lentivirus preparation Lentivirus preparations were produced by cotransfecting empty vector pLKO. 1puro with AXL shRNA, and helper virus packaging plasmids pCMV R8. 91 and pMD. G into 293T cells. Transfections were carried out using lipofectamine and PLUS reagent. Len tiviruses were harvested at 24, 36, 48, and 60 h post transfection. Virus was frozen at 80 C in appropriately sized aliquots for infection. Cell Culture and Virus infection OVCA429 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and seeded in six well plates.<br><br> Lentiviral shRNA infections were carried out in the presence of 8 ug mL polybrene. LY2228820 p38 MAPK 阻害剤 Cells were lysed for western blot analysis at 72 h post infection. Cell proliferation and apoptosis assays SKOV3, OVCA429, and ES2 cells were plated at 4, 000 cells well in a 96 well flat bottomed plate and cultured in media for 24 hours before being infected with lentiviral AXL shRNAs or different inhibitors, which included gefitinib, PHA 665752 alone or combination, 17 AAG, and AUY922, Cell viability and apoptosis were determined after treatment with inhibitors for 24 hours, and 3 and 6 days using the Caspase Glo 3 7 assay kit and the CellTiter Glo luminescent assay from Promega, and measured using a Veritas Microplate Luminometer, The data were normalized to the control group, All experimental points were set up in four replicate wells and independently performed in triplicate.<br><br> Apoptosis was also evaluated using PE Annexin V Apoptosis Detection Kit I, Briefly, SKOV3, OVCA429, and ES2 cells in 6 well plates were treated with 17 AAG or AUY922 for 48 hours, trypsinized and washed twice with cold Hanks Balanced Salt Solution and treated with 5 ul of PE Annexin V and 5 ul 7 AAD in 1X Binding Buffer for 15 minutes at RT in dark. The stained cells were analyzed in a flow cytometer within 1 hour and ModFit LT was used to analyze the data. Cell cycle analysis SKOV3, OVCA429, and ES2 cells in 6 well plates were treated with 17 AAG or AUY922 for 48 hours, then trypsinized and washed once with Hanks Balanced Salt Solution. For nuclear staining, cells were fixed by 70% ethanol for 24 h. A propidium iodide containing solution was added to the cells and incubated for 15 minutes at 37 C.