A present from John Lee, Smith Kline French) for 20 min before adherence. Genistein or SK F 86002 was dissolved in dimethyl sulfoxide (DMSO) and added towards the medium to a final concentration of 0.1 of DMSO. DMSO (0.1)does not interfere with mRNA stability or the mobility shift activity (data not shown). RNA isolation and Northern blot evaluation. Total RNA was isolated by the guanidine isothiocyanate-cesium chloride method (12). mRNA levels were determined by Northern blot evaluation. Total RNA (2 to 5 g per line) was electrophoresed on a 1 denaturing agarose gel and after that transferred to nitrocellulose (Schleicher Schuell) (38). Various blots have been carried out from each and every experiment, and all RNA levels have been equivalent according to 18S and 28S rRNA levels. Nitrocellulose blots have been probed with 32P-labeled cDNA probes made using a random-priming kit (Boehringer Mannheim). Hybridizations were incubated overnight inside a 50 (vol/vol) dimethylformamide solution at 42 . Blots had been washed with Coccidia site detergent at a final stringency of 0.2 SSPE (1 SSPE 0.18 M NaCl, ten mM phosphate [pH 7.4], 1 mM EDTA) at 56 and after that exposed to Kodak XAR2 X-ray film (Eastman Kodak) with intensifier screens at 70 . Northern blots probed with cDNAs for any with the three GRO-encoding genes, GRO , GRO , or GRO , cross-react to such an extent that selective identification needs PCR approaches (21); monocytes predominantly express GRO , so the hybridization information reflects GRO , but for accuracy it truly is labeled GRO. Cell extract preparation. For mobility shift assays, cell extracts have been prepared from nonadhered or adhered human monocytes as described previously (6) by lysis in 0.five ml of buffer A (ten mM Tris-HCl [pH 7.6], 1 mM magnesium acetate [Mg(OAc)2], 1.five mM KOAc, 2 mM dithiothreitol, 0.4 Nonidet P-40, 1 M phenylmethylsulfonyl fluoride, 1 M 1,10-phenanthrolene, antipain (50 g/ml), leupeptin (1 g/ml), pepstatin (1 g/ml), bestatin (40 g/ml), E64 (three g/ml), chymostatin (one hundred g/ml), and 10 glycerol). Each and every remedy group utilized 106 cells. Extracts were clarified by equivalent cell numbers, five 106 or 10 centrifugation (S20 postnuclear fraction). Alternatively, a cytosol S130 fraction was prepared as described previously (7). Both isolation strategies gave the identical results in gel shift CDK4 drug assays (data not shown). Within the present study, S20 extracts have been utilized for all experiments. Supernatants were collected and snap frozen on dry ice just before storage at 70 . Protein concentrations had been determined by the bicinchoninic acid approach (Pierce). Construction of plasmids and in vitro transcription. The BamHI-PvuII fragment of GRO , containing the GRO ARE, was cloned into BamHI-EcoRVdigested plasmid pcDNA1 such that transcription with SP6 RNA polymerase yielded sense RNA. The resulting plasmid p3 GRO was linearized with BamHI for the sense probe (BamHI 320 nt) or XmnI for the antisense RNA probe. In vitro transcription reactions have been performed with SP6 or T7 RNA polymerase (Promega), respectively. The BamHI 320 nt probe was utilized in all the gel shift experiments unless otherwise noted. A handle open reading frame (ORF) RNA probe (460 nucleotides [nt]) was produced by using T7 RNA polymerase and PvuIIdigested plasmid pcDNA1 GRO . The fragments of -globin and -globin plus (AUUU)five RNA, employed for competition experiments, had been created by in vitro transcription of plasmids p 19R and p 19R AT 5 linearized with SalI (40, 52). To decide if additional binding domains existed, RNA substrates were ready as follows (see Fig.
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