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1.
FIG. 8

FIG. 8. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Inability of LFB3Δ(400-450) to mediate cAMP-dependent induction of the ABC enhancer in LLC-PK1 cells. LLC-PK1 cells were transfected with pABC-TATA (0.5 μg) as luciferase reporter gene construct, along with increasing amounts (0, 50, and 100 ng) of empty vector or vectors expressing HALFB3Δ(400-450) or HALFB3. cAMP signaling was triggered by adding 1 mM Br-cAMP (6 h) before luciferase activity was measured.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.
2.
FIG. 5

FIG. 5. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Interaction of LFB3 with CREB and ATF1. Effects of the insertion of 5 or 10 nucleotides between domains A, B, and C on ABC enhancer-mediated cAMP inducibility. Luciferase constructs (1 μg) were induced either with 1 mM Br-cAMP or by cotransfecting 0.5 μg of pCEV, a vector expressing a catalytic subunit of the PKA. Assays were done in duplicate, and mean values are shown with error bars. For details of the different constructs, see Materials and Methods and Fig. 1A.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.
3.
FIG. 7

FIG. 7. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Unimportance of CREB Ser-133 in LFB3 binding. Two CREB mutants, a Ser-to-Ala mutant (CREBS133A) and a mutant lacking the leucine zipper (CREBΔ) (0.5 μg each), were cotransfected into LLC-PK1 cells together with a vector expressing Gal4-LFB3(315-559) (0.5 μg) and the CAT reporter gene construct pG5CAT (1 μg). cAMP signaling was triggered by coexpression of the catalytic subunit of PKA (pCEV, 0.5 μg). CAT activity was measured as described in Materials and Methods.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.
4.
FIG. 2

FIG. 2. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Specificity of LFB3 cooperation. LLC-PK1 cells were transfected with different reporter constructs and 16 h later induced either with 1 mM Br-cAMP or with 100 ng of TPA per ml for 6 h. Cells were then collected, and luciferase activity was measured. pAP1-TATA contains three copies of the AP1 sequence from the collagen gene. In the pAP1C-TATA construct, CRE-like sequences were converted to AP1. For details of these constructs, see Materials and Methods. Luciferase activity was measured as for Fig. 1C, and values are plotted as fold induction relative to noninduced cells.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.
5.
FIG. 4

FIG. 4. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Increased DNA-binding activity of nuclear extracts (NE) from LLC-PK1 cells by PKA treatment. Nuclear extracts were treated where indicated with 100 U of the catalytic subunit of PKA for 90 min at 37°C. Afterwards, samples were incubated with or without 2 μg of the indicated antibodies (Ab) for 4 h at 4°C and assayed for DNA-binding activity as described in the legend to Fig. 3. The gels were dried and exposed to X-Omat AR film for 1 day for the somatostatin CRE probe and 4 days for the domain B probe.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.
6.
FIG. 6

FIG. 6. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Analysis of the LFB3 molecule in a mammalian two-hybrid system for interaction with CREB and ATF1. (A) Schematic representation of Gal4-LFB3 and its deletion derivatives. DD, dimerization domain; POU, POU-specific domain; Pro-Glu, proline- and glutamine-rich activation domain. HA-tagged LFB3Δ(400-450) is diagrammed below. (B) CREB and ATF1 interact with the C terminus of LFB3. The CAT reporter gene construct pG5CAT (1 μg) was cotransfected into LLC-PK1 cells with vectors expressing different Gal4-LFB3 deletion mutants and with either pVP16, pVP16-CREB, or pVP16-ATF1 (1 μg each). cAMP signaling was triggered by coexpressing the catalytic subunit of PKA (pCEV, 0.5 μg). CAT activity was measured as described in Materials and Methods. (C) Mapping of the LFB3 site interacting with CREB and ATF1. The CAT reporter gene construct pG5CAT was cotransfected into LLC-PK1 cells with vectors expressing different Gal4-LFB3 deletion mutants and with either pVP16, pVP16-CREB, or pVP16-ATF1. cAMP signaling was triggered by coexpressing the catalytic subunit of PKA (pCEV). CAT activity was measured as described above.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.
7.
FIG. 1

FIG. 1. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Significance of the deviation of protein-binding sequences in the ABC enhancer from respective consensus sequences. (A) Wild-type and mutated sequences derived from the ABC enhancer located at −3.4 kb of the pig uPA promoter were cloned in front of a reporter gene. The mutated sequences are shown in lower case (in reverse type), the half-palindromic CREs are boxed, and domains A, B, and C are separated by vertical lines. (B) Different constructs were transiently transfected into LLC-PK1 cells; 16 h later, cells were induced by 1 mM Br-cAMP for 6 h and luciferase activity was measured. (C) pABC-TATA and pABHNF-TATA were transiently transfected into F9 cells with or without expression vectors for the catalytic subunit of PKA (CEV) and LFB3. After 16-h transfection, cells were collected and luciferase activity was measured.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.
8.
FIG. 9

FIG. 9. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Distinct functions of LFB3 for basal transactivation and mediation of cAMP induction. (A) LFB3Δ(400-450) retains its transactivation capacity. 293 cells were transiently transfected with the cAMP-independent reporter gene expression vector pAlb-Luc (0.5 μg) together with vectors encoding either HALFB3 or HALFB3Δ(400-450) (50 ng). Luciferase activity was measured and expressed as for Fig. 3B. (B) LFB3Δ(400-450) is unable to mediate cAMP induction. 293 cells were transiently transfected with the cAMP-dependent reporter gene expression vector pABC-TATA (0.5 μg) together with vectors encoding either HALFB3 or HALFB3Δ(400-450) (5 ng). cAMP signaling was triggered by coexpressing the catalytic subunit of PKA (pCEV, 0.5 μg). Luciferase activity was measured and expressed as described above. (C) CREB and ATF1 coprecipitate with LFB3 but not with LFB3Δ(400-450). In vitro [35S]methionine-labeled CREB or ATF1 was incubated with empty Ni-chelating Sepharose beads (Pharmacia) (control) or with beads coupled with His-tagged LFB3 or LFB3Δ(400-450) protein. After a wash and elution, samples were subjected to SDS-polyacrylamide gel electrophoresis. The bands were visualized by fluorography.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.
9.
FIG. 3

FIG. 3. From: Role of Tissue-Specific Transcription Factor LFB3 in a Cyclic AMP-Responsive Enhancer of the Urokinase-Type Plasminogen Activator Gene in LLC-PK1 Cells.

Involvement of ATF1 and CREB in ABC enhancer-mediated cAMP induction. (A) EMSA. Nuclear extracts (NE) prepared from LLC-PK1 cells were tested for binding to oligonucleotides containing sequences from the somatostatin promoter CRE region or from domain A or B. Radioactive oligonucleotides indicated at the bottom of each panel were incubated without (−) or with nuclear extracts plus anti-ATF1 (α-ATF1) antibody, anti-CREB antibody, nonspecific antiserum (immunoglobulin G) (ns. IgG), or a 100-fold excess of cold oligonucleotide of the same sequence. Reaction products were fractionated in nondenaturing acrylamide (5%) gel electrophoresis and exposed in a PhosphorImager or to X-Omat X-ray film with an intensifying screen at −70°C for 4 days for the A and B probes and 3 days for the somatostatin CRE probe. (B) Detection of CREB and ATF1 by blotting EMSA gels. Nuclear extracts (25 μg of protein) from LLC-PK1 cells were incubated in the absence of oligonucleotide (control) or with oligonucleotides corresponding to domain A or B or somatostatin CRE or a nonspecific oligonucleotide (ns) and separated by nondenaturing gel electrophoresis. Proteins were analyzed by immunoblotting with polyclonal antisera to CREB and ATF1. Only regions corresponding to the main complex are shown. (C) Comparisons of different cell lines for ABC enhancer-mediated cAMP induction. Cell lines were transfected with 1 μg of pABC-TATA together with 0.5 μg (or 0.1 μg for COS-1 and NIH 3T3 cells) of pRSV-LFB3 and/or 0.5 μg of pCEV. After 16-h transfection, cells were collected and luciferase activity was measured. Assays were done in duplicate, and mean values are shown with error bars.

Mazin Khalil Soubt, et al. Mol Cell Biol. 1998 August;18(8):4698-4706.

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