Lastly, the approach can turned out to be expensive if extended towards the ultra high throughput setting. We’ve got hence modified the approach to allow for the effective amplification of as much as a hundred transcripts using a ligation medi ated amplification approach, followed by detection on polysty rene beads by means of flow cytometry, as we not too long ago described. The current research, however, establishes the GE HTS notion will be utilized to screening for modulators of signal transduction, representing a basic technique to your discovery of compounds that impact any signaling pathway of curiosity. Mediatech, penicillin and streptomycin from Invitrogen, and fetal bovine serum from Sigma. p44/42 MAP kinase, phospho p44/42 MAP kinase, MEK1/2, phospho MEK1/2, PDGF BB, phos pho PDGFR, phospho EGFR, cKIT, Phospho cKIT, IGF I R, and Phospho IGF IRinsulin receptor antibodies have been obtained from Cell Signaling.
EGFR and mouse cKIT antibod ies were obtained from Santa Cruz Biotechnology. Alfa tubulin antibody was obtained from Sigma. Cells SH SY5Y neuroblastoma cells have been obtained from Ameri can Kind Culture Assortment. The IL3 dependent pro B lymphoid cell line Ba/F3 and Ba/F3 cells expressing TEL/ PDGFR had been buy PCI-32765 obtained from Dr. Gary Gilliland. TIP5 primary fibroblasts have been a gift from Dr. Stephen Less nick. We thank Dr. Ruth Halaban for 501 MEL human melanoma cells. PER expressing PC12 cells have been generously provided by Dr. Darren Tyson. SH SY5Y, PC12, TIP5 and MCF7 cells were cultured in DMEM, BaF3 cells and BaF3 cells expressing TEL/PDGFR have been maintained in RPMI 1640 medium, and 501 MEL cells have been grown in Hams ten medium. Medium for IL3 dependent Baf3 cells was supple mented with 0. 05 ng/ml IL3. Media for all cell lines except PC12 contained 10% fetal bovine serum, ten U/ml penicillin, and 10 g/ml streptomycin.
PC12 cells had been grown in DMEM with 15% horse serum, 5% fetal bovine serum, ten U/ml peni cillin, and 10 g/ml streptomycin. All cells had been grown at 37 C in 5% CO2. Characterization XL147 from the activation signature for ERK/ PDGFR pathway SH SY5Y cells had been grown to confluence and starved in excess of night in serum zero cost medium so as to silence any sustained results from growth factor signaling. Before induction with 50 ng/ml PDGF, cells were handled with pathway inhibitors 74M apigenin or 50M U0126, or with dimethyl sulfoxide compound remedy, cells had been induced with forty l of PDGF diluted in media. PDGF was added to half of control wells to measure PDGF response,only media was additional for the remaining handle wells. After forty minutes of PDGF induction, media was discarded, cells had been lysed and RNA was extracted and quantified as described in. Briefly, 15 l of lysis option containing a hypotonic deter gent, dithiothreitol frameborder=”0″ allowfullscreen> and RNAse inhibitor had been extra to medium zero cost cells for 15 minutes.