1 mM non essential amino acids. Trans fection was performed using the Lipofectamine 2000 reagent according to the manufacturers procedure. Stable cell lines were generated by infecting the cells twice with viral supernatants prepared from the 293T cells and colonies http://www.selleckchem.com/products/Trichostatin-A.html were established after selec tion using blasticidin for 72 hrs. The following small chemical inhibitors were used in this study in MCF 7 cells colchicine, cytochalasin B, wortmannin, monensin, rapamycin, staurosporine. siRNA treatment and real time RT PCR siRNAs to human SNX16 and SNX23 were designed and synthesized by Ribobio. The target sequences are Transfection of siRNAs was performed using the DharmFECT transfection re agent according to the manufacturers protocol and the final concentration of siRNAs was 50 nM.
The efficiency of siRNA was determined by real time RT PCR at 48 or 72 hrs post transfection. Briefly, total RNA was extracted from cells using the Trizol reagent. cDNAs were prepared from 5 ug of RNA with the ReverTra Ace Kit. Quantitative PCR was performed using the Inhibitors,Modulators,Libraries Premix Ex Taq and analyzed with CFX96 Touch Real Time PCR Detection System. Three independent assays were per formed for Inhibitors,Modulators,Libraries each sample and data represents mean SD. The primers used are. Immunofluorescence staining Cells on glass coverslips were fixed in 4% paraformalde hydePBS for 30 min, washed with 2 mgml glycinePBS for 5 min and permeabilized in 0. 2% Triton X 100PBS for 15 min. After two brief washes in PBS, cells were blocked in 3% NGSPBS for 1 hr at RT. Samples were then incubated in primary antibody for 1 hr at RT.
After four washes with 1% BSA0. 05% Tween 20PBS and three washes with PBS, cells were Inhibitors,Modulators,Libraries incubated in Alex 488 or 568 conjugated goat anti mouse or goat anti rabbit IgG secondary antibody for 1 hr. Cells were then washed four times with 1%BSA0. 05% Tween 20PBS and three times with PBS, counterstained with DAPI for 3 min and mounted. Mouse heart frozen sections were pre pared using freezing microtome. Sections on slides were fixed in ice acetone for 5 10 min, air dried and then washed with PBS for 10min. Immunofluorescence stain ing on sections were performed as described above. The anti SNX16 rabbit polyclonal antibody was home made in our lab and used at the 1 50 dilution. To test the speci ficity of the antibody, purified human SNX16 protein was used to block the staining.
Other primary antibodies used are mouse anti Flag and rabbit polyclonal anti Paxillin Images were obtained with the Leica SP2 confocal microscope. Cell migration assay Cell migration was assayed with the Cell Motility HCS Reagent Kit. Inhibitors,Modulators,Libraries Briefly, blue fluorescent micro sphere solution was added to 24 well plate coated with 1% gelatin. The plate was washed twice with the Wash Buffer Inhibitors,Modulators,Libraries after 1 hr incubation at 37 C in the dark. Cells were www.selleckchem.com/products/Tipifarnib(R115777).html seeded into the plate and moni tored every 2 hrs. Images were analyzed using the Image Pro Plus 5. 0 software.