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The human CL-1 promoter luciferase reporter construct was made by cloning the -517 to +63 nucleotide sequences of the 5′-flanking region of human CL-1 promoter, which contains a minimal promoter from human genomic DNA, by polymerase chain reaction-based cloning. A Hind III deletion site was engineered into the 3′ end of the promoter fragment. This fragment was then subcloned into the Kpn 1-Hind III site in PGL3 plasmid (Promega) to give the (-517 to +63) hCL-1/Luc construct. Transfected cells were lysed in luciferase lysis buffer (Promega), and 3 μg of protein as analyzed by the Bradford method was assayed for luciferase activity with a luminometer. Transfection efficiencies, determined by cotransfection with β-Gal construct (2 μg), were essentially similar.
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RSFs were fixed in 4% paraformaldehyde, permeabilized with Triton-X100, and then stained with a polyclonal antibody to the p65 (Rel) component of NF-κB (Santa Cruz Biotech) and with a fluorescein-conjugated antibody to rabbit Ig
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RSFs were fixed in 4% paraformaldehyde, permeabilized with Triton-X100, and then stained with a polyclonal antibody to the p65 (Rel) component of NF-κB (Santa Cruz Biotech) and with a fluorescein-conjugated antibody to rabbit Ig.
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Cell lysates (40 μg) were resolved by SDS-polyacrylamide gel electrophoresis 12% geO and immunoblotted with polyclonal to IκB antibody (Santa Cruz Biotech). Supershift analysis was done by incubating nuclear extracts (5 μg) with antibody to the p50 component of NF-κB (1 μg; Santa Cruz Biotech) for 30 min at ambient temperature
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Cell lysates (40 μg) were resolved by SDS-polyacrylamide gel electrophoresis (12% geO and immunoblotted with polyclonal to IκB antibody (Santa Cruz Biotech). Supershift analysis was done by incubating nuclear extracts (5 μg) with antibody to the p50 component of NF-κB (1 μg; Santa Cruz Biotech) for 30 min at ambient temperature.
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4 with a polyclonal antibody to rabbit IL-1α (Endogen)
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4 with a polyclonal antibody to rabbit IL-1α (Endogen).
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We thank C. Damsky for the gift of anti-α5 (BIIG2) and critical reading of the manuscript and D. Williams-Herman for assistance in preparing the hCL-1/ Luc construct. Supported by NIH grants DE10306 and AR20684 (Z.W.), a fellowship from the American Lung Association, a Mentored Clinician Scientist Award (HL03732) (F.K.), a Pew International Foundation Fellowship (E.W.), and an Institutional National Research Service Award (ES07106)
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We thank C. Damsky for the gift of anti-α5 (BIIG2) and critical reading of the manuscript and D. Williams-Herman for assistance in preparing the hCL-1/ Luc construct. Supported by NIH grants DE10306 and AR20684 (Z.W.), a fellowship from the American Lung Association, a Mentored Clinician Scientist Award (HL03732) (F.K.), a Pew International Foundation Fellowship (E.W.), and an Institutional National Research Service Award (ES07106).
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