Lastly, the strategy can grow to be costly if extended to the ultra higher throughput setting. We’ve therefore modified the method to permit for the efficient amplification of as much as a hundred transcripts using a ligation medi ated amplification system, followed by detection on polysty rene beads by means of flow cytometry, as we not long ago described. The present review, yet, establishes that the GE HTS concept may be applied to screening for modulators of signal transduction, representing a common method on the 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 have been obtained from Santa Cruz Biotechnology. Alfa tubulin antibody was obtained from Sigma. Cells SH SY5Y neuroblastoma cells were purchased from Ameri can Form Culture Assortment. The IL3 dependent professional B lymphoid cell line Ba/F3 and Ba/F3 cells expressing TEL/ PDGFR were selleck chemicals EGFR Inhibitors obtained from Dr. Gary Gilliland. TIP5 primary fibroblasts had been a present from Dr. Stephen Less nick. We thank Dr. Ruth Halaban for 501 MEL human melanoma cells. PER expressing PC12 cells had been generously presented by Dr. Darren Tyson. SH SY5Y, PC12, TIP5 and MCF7 cells have been cultured in DMEM, BaF3 cells and BaF3 cells expressing TEL/PDGFR have been maintained in RPMI 1640 medium, and 501 MEL cells were grown in Hams 10 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, 10 U/ml penicillin, and 10 g/ml streptomycin.
PC12 cells had been grown in DMEM with 15% horse serum, 5% fetal bovine serum, 10 U/ml peni cillin, and 10 g/ml streptomycin. All cells were grown at 37 C in 5% CO2. Characterization NSC-207895 of your activation signature for ERK/ PDGFR pathway SH SY5Y cells were grown to confluence and starved over night in serum cost-free medium in order to silence any sustained effects from development factor signaling. Just before induction with 50 ng/ml PDGF, cells had been treated with pathway inhibitors 74M apigenin or 50M U0126, or with dimethyl sulfoxide compound therapy, cells have been induced with 40 l of PDGF diluted in media. PDGF was additional to half of handle wells to measure PDGF response,only media was extra towards the remaining handle wells. Following 40 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 choice containing a hypotonic deter gent, dithiothreitol and RNAse inhibitor had been added to medium cost-free cells for 15 minutes.