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2, and 1 × reaction buffer (Boehringer Mannheim). The Fas primer pair, 5′-CCCAAATAGGAGTGTATGCAGAG-G-3′ and 5′-GCCATTAAGATGAGCACCAAGG-3′, generated a 547-base pair (bp) product. The Ice primer pair, 5′-TTGCTCCCTAGAAGAAGCTCAA-AG-3′ and 5′-GCCTTCCCGAATACCATGAGAC-3′, generated a 321 -bp product. The Cpp32 primer pair, 5′-TGGAACAAATGGACCTGTTGACC-3′ and 5′-AGGACTCAAATTCTGTTGCCACC-3′, generated a 365-bp product. The Ice/rel2 primer pair, 5′-CGCTGAGGGCATTTGCTACC-3′ and 5′-CACTTCCAAGGATGCTGGAGAG-3′, generated a 272-bp product. The lch-1 primer pair, 5′-TCCAGCTCCAAGAGGTTTTTCAG-3′ and 5′-GTCCCTTTGAGGCAGGCATAG-3′, generated a 250-bp product. The Mch2α primer pair, 5′-CTGTTAGCCACGCAGATGCC-3′ and 5′-TGTTCACCAGTGTGAGGAGTTCTG-3′, generated a 447-bp product. The Mch3 primer pair, 5′-GCTGAGAAGCAATGGGTCACTC-3′ and 5′-TGCAGTGG ACACAGCCATGAG-3′, generated a 300-bp product. The glyceraldehyde phosphate dehydrogenase (GAPDH) primer pair, 5′-CCATGGAGAAGGCTGGGGC-3′ and 5′-CCAAAGTTGTCATGGATGTCC-3′, generated a 195-bp product. All products were analyzed by 1.0% agarose gel electrophoresis.
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6 per 10-cm plate) were washed twice with ice-cold phosphate-buffered saline (PBS), scraped into PBS (1 ml), and sedimented in a microcentrifuge for 30 s at 4°C. The supernatant solution was removed, and the sedimented material was resuspended in 100 μl of lysis buffer (PBS, 0.1% phenylmethanesulfonylfluoride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and incubated on ice for 20 min. The extracts were sedimented in a microcentrifuge for 30 min at 4°C. The supernatant solution represented whole-cell extract; 100 μg of this protein was analyzed by SDS-polyacrylamide gel electrophoresis (10% gel) and subsequently transferred to a polyvinylidine difluoride membrane for 12 hours at 4°C. The membrane was incubated for 12 hours in 5% blotto {5% Carnation milk in 1 × tris-buffered saline (TBS) [10 mM tris-HCI (pH 8.0) and 150 mM NaCl]}. Monoclonal antibodies to lch-1, Cpp32, and Fas (Signal Transduction Laboratories) were added to separate transfers at 1:1000 dilutions, and a polyclonal rabbit antiserum to Ice (Santa Cruz Biotech, Santa Cruz, CA) was added at 1 μg/ml. After incubation at room temperature for 2 hours, tne transfers were washed three times with 1 × TBS for a total of 30 min. Secondary antibodies were incubated with the transfers at 1:1000 dilutions for 1 hour at room temperature. The transfers were washed four times with 1 × TBS for a total of 1 hour and analyzed by enhanced chemiluminescence using Pierce reagents, followed by exposure to x-ray film.
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In these experiments, we were not able to analyze Ice protein, which is present at a very low level, with the antibody preparations available to us.
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Supported by grant P01 CA62220 from NIH. Thanks to S. Der, B. R. G. Williams, S. Bandyopadhyay, S. Leung, X. Li, D. Leaman, and J. Tebo for helpful comments and to J. Lang for photography.
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