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Unformatted text preview: Xba I (sites indicated by lowercase letters in the primers) and cloned into the Bam HI and Xba I sites of the vector pSK–. Plasmid pTNF- a 1281-1350 con- tained the seven AUUUA motifs of the TNF- a ARE (bases 1281 to 1350 of X02611). This was construct- ed using similar methods. Correct sequences of these plasmids were confirmed by dideoxy sequencing (Amersham). For radiolabeling of the RNA transcripts with [ a- 32 P]uridine triphosphate (800 Ci/mmol), plasmid TNF- a 1197-1350 was linearized with Xba I and used as the template in the Riboprobe in vitro transcription system (Promega) protocol. The result- ing product was precipitated with ammonium ace- tate and ethanol. 27. J. Y. Tso, X. H. Sun, T. H. Kao, K. S. Reece, R. Wu, Nucleic Acids Res. 13 , 2485 (1985). 28. Confluent dishes were washed three times with cys- teine-free medium supplemented with fetal calf se- rum (10%). Cells were stimulated for 4 hours in the same medium with control conditions, LPS (1 m g/ml), or TNF- a (10 ng/ml). For the last 3 hours of the incubation, [ 35 S]cysteine (200 m Ci/ml; NEN Life Sci- ences, Boston) was added to the cultures. Cells were washed twice with ice-cold PBS, scraped into 10 ml of PBS, and pelleted by centrifugation (1000 g for 5 min at 4°C). Cells were then resuspended in 600 m l of lysis buffer [50 mM tris-HCl (pH 7.5), 50 mM NaCl, 3 mM MgCl 2 , 5% (v/v) glycerol, 0.5% (v/v) NP-40, 0.02% (w/v) sodium azide, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), soybean tryp- sin inhibitor (20 m g/ml), and leupeptin (8 m g/ml)], incubated on ice for 20 min, and lysed by passage five times through a 28-gauge needle attached to a 1-ml syringe with no dead space (Becton Dickinson). The nuclear pellet (after centrifugation at 1000 g for 5 min at 4°C) was washed once in ice-cold wash buffer [10 mM tris-HCl (pH 7.5), 15 mM KCl, 1.5 mM MgCl 2 , 0.5 mM PMSF, and 5% glycerol], centrifuged at 1000 g for 5 min at 4°C, and then resuspended and sonicated in the same volume of lysis buffer used initially to lyse the cells. The cytosolic fraction (su- pernatant) was clarified by centrifugation at 45,000 g for 30 min at 4°C, using a tabletop ultracentrifuge (Beckman TL-100, rotor TLA.45). This method results in separation of cytosol and nuclear fractions, as assessed by protein immunoblotting with an anti- body to SP1 as described ( 18 ). Cytosolic extracts matched by trichloroacetic acid–precipitable radioac- tivity and equivalent volumes of nuclear extracts were incubated with preimmune rabbit serum (1:100 dilution, 1 hour at 4°C) and protein A–Sepharose (1 hour at 4°C), and then incubated overnight at 4°C in the presence of either preimmune serum (1:100) or a 1:100 dilution of a polyclonal rabbit antibody to mouse TTP ( 18, 19 ). Immune complexes were recov- ered by centrifugation after the addition of protein A–Sepharose, washed three times with wash buffer [50 mM tris-HCl (pH 8.3), 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40], resuspended in 100 m l of SDS...
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This note was uploaded on 10/07/2009 for the course GEN 409 taught by Professor Linda during the Fall '09 term at Iowa State.
- Fall '09