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note
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Histidine-tagged dMyc and dMax proteins were expressed from pET19b vectors (Novagen) containing a dMyc cDNA fragment (amino acids 534 to 717) or a dMax cDNA fragment (amino acids 26 to 161), and purified. In vitro transcription and translation were performed as in (4) using pRc/CMV vectors (Invitrogen, San Diego, CA). For in vitro interaction assays, 4 μl of each translated protein was incubated with 2 μl of His-dMyc or His-dMax attached to a nickel resin or 2 μl of bovine serum albumin (BSA)-blocked nickel resin in 400 μl of L buffer (phosphate-buffered saline containing 0.4% NP-40) for 1 hour at 4°C. After three washes with L buffer, the bound proteins were eluted with SDS-containing sample buffer and subjected to SDS-PAGE and autoradiography.
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41
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10544243594
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note
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2, 0.5 mM EDTA, 5% glycerol, 10 mM dithiothreitol, 0.1% NP-40, 0.5 mg/ml BSA, and 1 μg of sheared salmon sperm DNA for 30 min at room temperature. For competition experiments, increasing amounts of unlabeled CM-1 or B1/B2 (4) oligonucleotide were included in the binding reaction. The DNA-protein complexes were resolved on a 4% polyacrylamide gel containing 2.5% glycerol and subjected to autoradiography.
-
-
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42
-
-
10544256816
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Wiley, New York
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5 per 6-cm dish) were transfected with 1 μg of CMV-βgal, 2 μg of pGL2M4 [the vector pGL2 (Promega, Madison, WI) containing a fourfold reiteration of the sequence CACGTG] and the indicated amount of pRc/ CMV-dMyc, pRc/CMV-dMax, and pRc/CMV vector to a total of 9 μg. After 48 hours, luciferase activity was determined [F. M. Ausubel et al., Eds., Current Protocols in Molecular Biology (Wiley, New York, 1995) Supplement 29, pp. 9.7.11-9.7.21] and normalized for expression from CMV-βgal. The data represent duplicate samples from two independent experiments.
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Polyadenylated [poly(A)'] RNA was analyzed as in S. M. Parkhurst and V. G. Corces [Cell 41, 429 (1985)]. For the developmental analysis shown in Fig. 3, wild-type flies were kept at 25°C.
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1-containing lines were obtained from the Bloomington stock center.
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note
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We thank N. H. Brown, R. Davis, J. C. J. Eeken, S. Elledge, J. Kiger, C. D. Laherty, A. P. Mahowald, M. Meyer, G. Poortinga, J. W. Tamkun, C. Thummel, P. P. Tolias, M. Watanabe, and the Bloomington Stock Center for reagents; D. Gottschling, S. Henikoff, T. Neufeld, and J. Roberts for critical readings of the manuscript; P. Goodwin for help with image analysis; J. Torgerson for assistance with the manuscript; and members of the Parkhurst, Eisenman, and Edgar laboratories for advice and support. Funded by National Institutes of Health-National Cancer Institute (NCI) grants RO1CA57138 (to R.N.E.), and RO1GM47852 (to S.M.P.), a Postdoctoral Fellow-ship from the Fonds National Suisse (to P.G.) and an NCI Japanese Foundation for Cancer Research Training Program in the U.S.-Japan Cooperative Cancer Committee (to Y.S.).
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