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note
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AC. elegans cDNA library fused to the GAL4 activating domain of the pACT vector (a gift of Robert Barstead) was screened with the HF7c yeast reporter strain for proteins that interact with CED-4S (22). A bait plasmid expressing ced-4S in pGBT-8 was constructed by polymerase chain reaction (PCR) amplification of plasmid cDNA to incorporate restriction sites, followed by ligation of the amplified DNA fragment in-frame with the GAL4 DNA-binding domain of pGBT-8. Positive library plasmids were selectively recovered from bacteria colonies by growth in media lacking leucine. cDNA inserts in the plasmid were characterized by restriction enzyme mapping and nucleotide sequence analysis on an automated DNA sequencer (Applied Biosystems model 373A). Plasmids encoding ced-9 mutants in pGBT-8 were generated by PCR amplification of ced-9 cDNA template, using 3′ primers that included the natural translation termination sequences as described (4, 5). Authenticity of all constructs was confirmed by dideoxy sequencing.
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16
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15644374076
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D. Wu, H. D. Wallen, G. Nuñez, data not shown
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D. Wu, H. D. Wallen, G. Nuñez, data not shown.
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17
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15644362212
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note
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6 293T cells with amounts (indicated in figure legends) of plasmid DNA by the calcium phosphate method. The expression of HA-CED-9, HA-CED-9ATM, and Myc-CED-4 was determined in total lysates by immunoblotting (23). For immunoprecipitations, cells were lysed in Nonidet P-40 isotonic lysis buffer (23) at 24 hours after transfection, and soluble lysates were incubated with either 1 μg/ml anti-HA (clone 12CA5, Boehringer Mannheim); 1 μg/ml rabbit anti-Myc (Santa Cruz), anti-FLAG (clone M2, Scientific Imaging Systems), or isotype-matched mouse IgG (anti-HA control); or normal rabbit serum (anti-Myc control) overnight at 4°C. Protein A-Sepharose 4B (Zymed Laboratories) was added [5% (v/v)] for an additional hour of incubation by rotation. Immune complexes were centrifuged and washed with excess cold Nonidet P-40 isotonic lysis buffer at least four times, separated on a 15% SDS-polyacrylamide gel, and immunoblotted with rabbit anti-Myc, anti-HA or anti-FLAG. The proteins were detected with an ECL system (Amersham).
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15644378155
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note
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293T cells were transfected with pcDNA3-HA-ced-9, pcDNA3-HA-ced-9ΔTM, pcDNA3-Myc-ced/-4S, or pcDNA3-Flag-gata-1 as described (13). At 24 hours after transfection, cells were incubated with anti-HA, anti-Myc (clone 9E10, Santa Cruz), anti-FLAG, or control mouse IgG for 1 hour at 23°C, and the labeling was visualized with fluorescein-conjugated goat antimouse IgG. After washing, the cells were mounted in Slowfade (Molecular Probes) and examined with a Bio-Rad MRC 600 scanning confocal microscope equipped with an argon-xenon laser (24).
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20
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15644362331
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unpublished results
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D. Wu and G. Nuñez, unpublished results.
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Wu, D.1
Nuñez, G.2
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21
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15644364985
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note
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7) were transiently transfected with the indicated expression plasmids (10 μg). The cells were washed twice with phosphate-buffered saline and then incubated for 10 min in 1 ml of cold hypotonic buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 p.g antipain, and leupeptin (0.3 μg/ml) as described (25). The swollen cells were lysed by 15 strokes in a tight-fitting Douncer homogenizer, and the samples were centrifuged at 1000g for 6 min to remove nuclei. The resulting supernatant was centrifuged for 1 hour at 100,000g. The supernatant portion of the latter centrifugation was the soluble cytosolic (C) fraction; the pellet was the membrane and cytoplasmic organelle (M) fraction. The proteins from the C and M fractions were dissolved in 1 ml of lysis buffer, and samples of equal size were analyzed by immunoblotting.
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22
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0027525536
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0027225445
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15644384152
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note
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We thank R. Horvitz for plasmids and R. Barstead for the two-hybrid C. elegans cDNA library; C. Edwards and B. Donohoe for expert advice in confocal microscopy; M. Clarke and R. Ellis for stimulating discussions; and M. Benedict, P. Simonian, D. Ekhterae, M. González-García, Y. Hu, and N. Inohara for critical review of the manuscript. Supported in part by NIH grant CA-64556, Immunopathology NIH predoctoral training grant T32A107413-03 (H.D.W.), and an NIH Research Career Development Award (G.N.).
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