1. Transcriptional Control of the Mouse Col7a1 Gene in Keratinocytes: Basal and Transforming Growth Factor-β Regulated Expression 
Michael Naso, Jouni Uitto, John F. Klement 
Journal of Investigative Dermatology 
Volume 121, Issue 6, Pages (December 2003)
DOI: /j x
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Western blot analysis of proteins expressed by spontaneously transformed MKC cultures (K) and in wild-type, day 2 neonatal mouse skins (S). Molecular weight (MW) markers are in kDa. Antibodies used to visualize proteins on the blots are listed under the appropriate lanes.
Journal of Investigative Dermatology  , DOI: ( /j x)
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3. Figure 2
Primer extension analysis of the transcription start site of the Col7a1 gene. L indicates the nucleotide ladder used for size markers; MK (lane 1) represents RNA from the MKC culture, and Y (lane 2) represents control RNA from yeast cells. Nucleotide ladder fragment lengths are indicated by the numbers on the left. The arrow on the autoradiogram indicates the primer extended product.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Comparison of the 5′ flanking and UTR of the human and mouse type VII collagen genes. (A) The upper sequence is mouse and the lower sequence is human. Vertical lines indicate bases of identity between the sequences and gaps in the sequences are denoted by a series of periods. Putative transcription factor binding sites are boxed and labeled underneath the box. The arrows indicate the transcription start sites. (B) Locations of the Col7a1 promoter regions used for EMSA (boxed sequences). Mutations introduced into the Col7a1 promoter CAT constructs are depicted as lowercase letters above the sequence. The transcription factor binding sites are underlined and labeled accordingly beneath the site.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Results from CAT assays used to detect the activity of various Col7a1 promoter–CAT constructs transiently transfected into MKC cultures and schematic representation of each construct. The data are presented as a percentage of the construct generating the most CAT activity (–560). Bars extending to the right represent the mean±SEM of four experiments each done in duplicate. Numbers on the left in the schematic figure refer to the position (basepairs) of the 5′ end of the Col7a1 flanking sequence included in each construct. The top line (designated as 1) represents the CAT vector without a promoter.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
CAT activity of constructs with mutations in consensus transcription factor binding sites (indicated by arrowheads and labeled on the left) in Col7a1 promoter CAT constructs transiently transfected into MKC cultures. Data are presented as a percentage of the wild-type (WT) p-560Col7a1/CAT construct. Bars on the right represent the mean±SEM of four experiments each done in duplicate.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
EMSA depicting keratinocyte nuclear proteins binding to the –136/–97 double-stranded oligonucleotide of the Col7a1 promoter. E denotes lanes where keratinocyte nuclear extract was used in the EMSA (lanes 2–6). EMSA without nuclear extract is denoted as (–) (lane 1). “COLD” refers to unlabeled –136/–97 oligonucleotide (lane 3). “Sp1 Con” refers to unlabeled double-stranded oligonucleotides containing a consensus Sp1 DNA binding site (lane 6). Supershift EMSA containing anti-Sp1 and anti-Sp3 antibodies are denoted by α-Sp1 and α-Sp3, respectively (lanes 4, 5). Arrows point to complexes that can be disrupted by consensus oligonucleotides, and arrowheads indicate DNA–protein–antibody complexes supershifted with antibodies to Sp1 and Sp3.
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
EMSA depicting the binding of keratinocyte nuclear proteins from the keratinocyte cell line to double-stranded oligonucleotides contained in the distal region from –560 to –340 of the Col7a1 promoter. Oligonucleotides used in the EMSA spanning the –435/–409 sequence, the –474/–448 sequence, the –570/–538 sequence, or –539/–509 sequence are indicated above the lanes. Protein–DNA complexes are denoted by arrows and protein–DNA–antibody (supershift) complexes are denoted by arrowheads. Other abbreviations are as in Figure 6 and antibodies to AP1 and AP2 are denoted α-AP1 and α-AP2, respectively.
Journal of Investigative Dermatology  , DOI: ( /j x)
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9. Figure 8
Effects of over-expression of c-jun, Smad7, or dnSmad3 on the p-560Col7a1/CAT promoter activity in keratinocytes. Increasing amounts of each expression plasmid (100, 200, 400, and 800 ng), as depicted under the appropriate bars in the graph and labeled with the expression plasmid, were cotransfected with a constant amount of the p-560Col7a1/CAT plasmid (200 ng). Also, the expression vector without a cDNA insert (pcDNA) was used as a control in cotransfections. The values are expressed as the mean of three separate experiments each done in duplicate (±SEM).
Journal of Investigative Dermatology  , DOI: ( /j x)
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10. Figure 9
EFFECT of TG-β on CAT expression from mouse Col7a1 promotor constructs in MKC cultures. For (A) and (B), MKC cultures were transfected with Col7a1 promoter CAT constructs and then incubated for 3 h in medium without BPE. The cultures were divided into two equivalent sets. One set of transfections was then incubated with 5 ng per ml of TGF-β for 40 h and the corresponding set was incubated without TGF-β. The values are presented as the mean of four separate experiments each done in duplicate (± SEM). Fold induction is calculated for each plasmid as the CAT activity with TGF-β divided by the CAT activity without TGF-β. Transfections performed (A) with the Col7a1 5′ construct series, and (B) with p-560Col7a1/CAT mutant transcription factor binding site series. Labeling and designations are as in Figure 4 and Figure 5.
Journal of Investigative Dermatology  , DOI: ( /j x)
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11. Figure 10
Role of SMADs in TGF-β induction of mouse Col7a1 promoter in MKC cultures. (A) Levels of Smad2 and Smad3 in keratinocyte nuclear extracts after treatment with TGF-β as detected by western blotting. Western blots were performed with 10 μg of keratinocyte nuclear extract from cells treated with TGF-β (5 ng per ml) for 0, 5, 15, 30, and 60 min as indicated above the lanes, and probed with an antibody that detects both Smad2 and Smad3. The Smad2 or Smad3 protein bands are indicated by arrows and labeled. Note the rapid increase in Smad3 protein between 0 min and 5 min. (B) Effect of coexpression of c-jun, Smad7, and dnSmad3 on TGF-β induction of the p-560Col7a1/CAT promoter. The bar labeled “Control” represents cotransfection performed using the expression vector without a cDNA insert. Cells were cotransfected with p-560Col7a1/CAT construct and expression plasmids indicated, incubated in mKGM without BPE, and then treated with TGF-β (5 ng per ml) for 40 h in mKGM without BPE. The values are the mean of three separate experiments each done in duplicate (±SEM).
Journal of Investigative Dermatology  , DOI: ( /j x)
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