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Transcriptional activity of the collagen, type I, α1 (COL1A1) proximal promoter in normal and nephrogenic systemic fibrosis (NSF) fibroblasts. (A) Two COL1A1 promoter constructs (−804 and −174 bp) ligated to the bacterial chloramphenicol acetyl transferase (CAT) gene were transiently transfected into subconfluent monolayer cultures of normal (N) or NSF dermal fibroblasts. CAT activity was assayed after transfection in <t>TranSignal</t> whole cell lysates. The data are the average of duplicate assays. (B, C) Protein/DNA binding activities identified using the protein/DNA arrays and assessment of cREL binding activity in normal and NSF fibroblasts. (B) Array 1; (C) Array 2. Differential binding activity (cREL, NF1 and CBF) is underlined. The results shown are representative of two separate experiments, each performed in duplicate. (D) Electrophoretic mobility shift assay of DNA/protein complexes obtained with nuclear extracts from NSF or N fibroblasts incubated with cREL oligonucleotide probes. Lane 1: control (CNT; 100-fold unlabelled cREL oligonucleotide). Lanes 2–4: nuclear extracts from each of the NSF cell lines. Lanes 5 and 6: Nuclear extracts from each of the normal cell lines.
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Transcriptional activity of the collagen, type I, α1 (COL1A1) proximal promoter in normal and nephrogenic systemic fibrosis (NSF) fibroblasts. (A) Two COL1A1 promoter constructs (−804 and −174 bp) ligated to the bacterial chloramphenicol acetyl transferase (CAT) gene were transiently transfected into subconfluent monolayer cultures of normal (N) or NSF dermal fibroblasts. CAT activity was assayed after transfection in TranSignal whole cell lysates. The data are the average of duplicate assays. (B, C) Protein/DNA binding activities identified using the protein/DNA arrays and assessment of cREL binding activity in normal and NSF fibroblasts. (B) Array 1; (C) Array 2. Differential binding activity (cREL, NF1 and CBF) is underlined. The results shown are representative of two separate experiments, each performed in duplicate. (D) Electrophoretic mobility shift assay of DNA/protein complexes obtained with nuclear extracts from NSF or N fibroblasts incubated with cREL oligonucleotide probes. Lane 1: control (CNT; 100-fold unlabelled cREL oligonucleotide). Lanes 2–4: nuclear extracts from each of the NSF cell lines. Lanes 5 and 6: Nuclear extracts from each of the normal cell lines.

Journal: Annals of the rheumatic diseases

Article Title: Persistent activation of dermal fibroblasts from patients with gadolinium-associated nephrogenic systemic fibrosis

doi: 10.1136/ard.2009.127761

Figure Lengend Snippet: Transcriptional activity of the collagen, type I, α1 (COL1A1) proximal promoter in normal and nephrogenic systemic fibrosis (NSF) fibroblasts. (A) Two COL1A1 promoter constructs (−804 and −174 bp) ligated to the bacterial chloramphenicol acetyl transferase (CAT) gene were transiently transfected into subconfluent monolayer cultures of normal (N) or NSF dermal fibroblasts. CAT activity was assayed after transfection in TranSignal whole cell lysates. The data are the average of duplicate assays. (B, C) Protein/DNA binding activities identified using the protein/DNA arrays and assessment of cREL binding activity in normal and NSF fibroblasts. (B) Array 1; (C) Array 2. Differential binding activity (cREL, NF1 and CBF) is underlined. The results shown are representative of two separate experiments, each performed in duplicate. (D) Electrophoretic mobility shift assay of DNA/protein complexes obtained with nuclear extracts from NSF or N fibroblasts incubated with cREL oligonucleotide probes. Lane 1: control (CNT; 100-fold unlabelled cREL oligonucleotide). Lanes 2–4: nuclear extracts from each of the NSF cell lines. Lanes 5 and 6: Nuclear extracts from each of the normal cell lines.

Article Snippet: Nuclear extracts were subjected to the TranSignal Protein/DNA arrays I and II (Panomics, Redwood City, California, USA) as described.

Techniques: Activity Assay, Construct, Chloramphenicol Acetyltransferase Assay, Transfection, Binding Assay, Electrophoretic Mobility Shift Assay, Incubation, Control