Journal: Inflammopharmacology

Article Title: Herbal remedies for oral and dental health: a comprehensive review of their multifaceted mechanisms including antimicrobial, anti-inflammatory, and antioxidant pathways

doi: 10.1007/s10787-024-01631-8

Figure Lengend Snippet: Efficacy of herbal remedies against periodontal pathogens

Article Snippet: Lippia alba (Mill.) Cymbopogon citratus L. , Whole plant/essential oil extraction through steam distillation , An assay called MBEC-high-throughput (MBEC-HTP) to measure the amount of S. mutans ATCC 35668 strain biofilms that were eradicated CHO cells were subjected to cytotoxicity assessment using the MTT cell proliferation assay , Geraniol and citral were the main constituents of both oils; they were 18.9% and 15.9% in L. alba and 31.3% and 26.7% in C. citratus , respectively. No concentration of either essential oil was toxic to CHO cells over a 24-h period. The essential oils of L. alba and C. citratus demonstrated eradication activity against Streptococcus mutans biofilms of 95.8% and 95.4%, respectively, at 0.1 and 0.01 mg/dL concentrations and 93.1%, at 0.001 mg/dL concentration , (Tofiño-Rivera et al. ) .

Techniques: Diffusion-based Assay, Produced, Inhibition, Positive Control, Bacteria, Microbial Assay, In Vivo, Cytotoxicity Assay, Activity Assay, In Vitro, Derivative Assay, Concentration Assay, Time-Kill Assay, Extraction, Microdilution Assay, Sequencing, Expressing, Electron Microscopy, Confocal Laser Scanning Microscopy, MTT Assay, Enzyme-linked Immunosorbent Assay, Control, Functional Assay, Serial Dilution, Adsorption, Probiotics, Formulation, Comparison, Cannabis, Ex Vivo, Maltodextrin, Modification, Biofilm Production Assay, Permeability, Incubation, Membrane, Tube Formation Assay, Binding Assay, Migration, In Silico, Cell Counting, Recombinant, WST-1 Assay, Luminescence Assay, DNA Synthesis, Gene Expression, Irradiation, Serial Time-encoded Amplified Microscopy, Distillation, MTT Cell Proliferation, Sampling, Staining, Resazurin Assay, Solvent, Serial Dilution Assay, Dilution Assay, Antioxidant Activity Assay, Animal Model, Real-time Polymerase Chain Reaction, Isolation, Transmission Assay, Preserving, Colorimetric Assay, Saline, Activation Assay, Molecular Weight, Disruption, Labeling, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Novel Assays to Distinguish Between Properdin-Dependent and Properdin-Independent C3 Nephritic Factors Provide Insight Into Properdin-Inhibiting Therapy

doi: 10.3389/fimmu.2019.01350

Figure Lengend Snippet: Two approaches to distinguish between properdin-dependent and properdin-independent C3 nephritic factors (C3NeFs). In the first approach, rabbit erythrocytes (RbE) are incubated with properdin-depleted normal human serum (ΔP-NHS) substituted with an equal volume of patient immunoglobulins (Igs). In the second approach, properdin function is eliminated by adding the properdin-blocking tick protein Salp20 to patient serum, that is always mixed 1:1 with pooled normal human serum (NHS) to compensate for possible low C3 levels. All incubations in the first step take place under C5 inhibition by eculizumab to prevent complement activation up to the level of the C5b-9 complex formation and subsequent hemolysis. After a washing step, guinea pig serum in presence of EDTA is added in the second step of the assay as a source of C5b-9 components to read out the activity of preformed convertase complexes of the first step. Hemolysis is measured over time to form convertase activity profiles. Prolonged convertase activity in absence of properdin indicates increased convertase stabilization by properdin-independent C3NeFs, whereas normal convertase stability indicates that the C3NeFs are properdin-dependent.

Article Snippet: A DNA fragment encoding Ixodes scapularis Salp20 [UniProtKB: Q95WZ1 (residue 22–183)] was amplified by PCR from Salp20 synthetic DNA optimized for mammalian expression (GeneART ThermoFisher), and ligated into BamHI-NotI sites of vector pUPE106.03 (U-Protein Express BV, Utrecht, the Netherlands).

Techniques: Incubation, Blocking Assay, Inhibition, Activation Assay, Activity Assay

Journal: Frontiers in Immunology

Article Title: Novel Assays to Distinguish Between Properdin-Dependent and Properdin-Independent C3 Nephritic Factors Provide Insight Into Properdin-Inhibiting Therapy

doi: 10.3389/fimmu.2019.01350

Figure Lengend Snippet: Properdin depletion or blockage in normal human serum results in delayed maximal convertase activity. (A) Rabbit erythrocytes were incubated with properdin-depleted normal human serum (ΔP-NHS) and increasing concentrations of purified properdin (P). (B,C) Alternatively, erythrocytes were incubated with pooled normal human serum (NHS) and increasing concentrations of Salp20. Protein concentrations indicated are corrected for the used serum percentages of 3.8 (A) and 5.0% (B,C) . Error bars indicate standard deviations of the mean obtained from three independent experiments. hi-NHS, heat-inactivated NHS.

Article Snippet: A DNA fragment encoding Ixodes scapularis Salp20 [UniProtKB: Q95WZ1 (residue 22–183)] was amplified by PCR from Salp20 synthetic DNA optimized for mammalian expression (GeneART ThermoFisher), and ligated into BamHI-NotI sites of vector pUPE106.03 (U-Protein Express BV, Utrecht, the Netherlands).

Techniques: Activity Assay, Incubation, Purification

Journal: Frontiers in Immunology

Article Title: Novel Assays to Distinguish Between Properdin-Dependent and Properdin-Independent C3 Nephritic Factors Provide Insight Into Properdin-Inhibiting Therapy

doi: 10.3389/fimmu.2019.01350

Figure Lengend Snippet: Detection of properdin-dependent and properdin-independent C3NeFs in C3G patients using patient serum and the properdin inhibitor Salp20. Rabbit erythrocytes were incubated with pooled normal human serum (NHS) mixed with an equal volume of serum of patients P1, P2, P3, P4, and P6 or EDTA-plasma of P5 to a final concentration of 5% and in presence of 6.25 μg/ml Salp20. Additionally, purified C3 was added for the samples of P1, P3, and P4, according to the concentrations indicated, which are corrected for the used serum percentage. Error bars indicate standard deviations of the mean obtained from three independent experiments. ΔP-NHS, properdin-depleted normal human serum; hi-NHS, heat-inactivated NHS.

Article Snippet: A DNA fragment encoding Ixodes scapularis Salp20 [UniProtKB: Q95WZ1 (residue 22–183)] was amplified by PCR from Salp20 synthetic DNA optimized for mammalian expression (GeneART ThermoFisher), and ligated into BamHI-NotI sites of vector pUPE106.03 (U-Protein Express BV, Utrecht, the Netherlands).

Techniques: Incubation, Clinical Proteomics, Concentration Assay, Purification

Journal: Frontiers in Immunology

Article Title: Novel Assays to Distinguish Between Properdin-Dependent and Properdin-Independent C3 Nephritic Factors Provide Insight Into Properdin-Inhibiting Therapy

doi: 10.3389/fimmu.2019.01350

Figure Lengend Snippet: Effect of low serum C3 concentration on the convertase activity profile of serum treated with Salp20. (A) Rabbit erythrocytes were incubated with C3-depleted normal human serum (ΔC3-NHS) mixed with an equal volume of pooled normal human serum (NHS) in absence or presence of 6.25 μg/ml Salp20. (B) Alternatively, ΔC3-NHS samples treated with Salp20 were supplemented with increasing concentrations of purified C3. Concentrations indicated are corrected for the used serum percentages of 5.0 (A) and 3.8% (B) . Representative data of at least two independent experiments are shown. ΔP-NHS, properdin-depleted normal human serum; hi-NHS, heat-inactivated NHS.

Article Snippet: A DNA fragment encoding Ixodes scapularis Salp20 [UniProtKB: Q95WZ1 (residue 22–183)] was amplified by PCR from Salp20 synthetic DNA optimized for mammalian expression (GeneART ThermoFisher), and ligated into BamHI-NotI sites of vector pUPE106.03 (U-Protein Express BV, Utrecht, the Netherlands).

Techniques: Concentration Assay, Activity Assay, Incubation, Purification

Journal: Frontiers in Immunology

Article Title: Novel Assays to Distinguish Between Properdin-Dependent and Properdin-Independent C3 Nephritic Factors Provide Insight Into Properdin-Inhibiting Therapy

doi: 10.3389/fimmu.2019.01350

Figure Lengend Snippet: Convertase-stabilizing activity of properdin-dependent but not properdin-independent C3NeFs is restored in presence of functional properdin. Patient immunoglobulins (Igs) of P4 (A) and P1 (C) were incubated with properdin-depleted normal human serum (ΔP-NHS), and were reconstituted with increasing concentrations of purified properdin (P). Alternatively, Salp20 was titrated into the patient sera of patient P4 (B) and P2 (D) , that were tested mixed with an equal volume of pooled normal human serum (NHS). Protein concentrations indicated are corrected for the used serum percentage of 5%. Representative data of at least two independent experiments are shown. hi-NHS, heat-inactivated NHS.

Article Snippet: A DNA fragment encoding Ixodes scapularis Salp20 [UniProtKB: Q95WZ1 (residue 22–183)] was amplified by PCR from Salp20 synthetic DNA optimized for mammalian expression (GeneART ThermoFisher), and ligated into BamHI-NotI sites of vector pUPE106.03 (U-Protein Express BV, Utrecht, the Netherlands).

Techniques: Activity Assay, Functional Assay, Incubation, Purification

Journal: Frontiers in Immunology

Article Title: Novel Assays to Distinguish Between Properdin-Dependent and Properdin-Independent C3 Nephritic Factors Provide Insight Into Properdin-Inhibiting Therapy

doi: 10.3389/fimmu.2019.01350

Figure Lengend Snippet: Screening for properdin-dependent C3NeFs in patient sera using the Salp20 method. Convertase activity was assessed in the serum of patients P7–P13 mixed with an equal volume of pooled normal human serum (NHS) to a final percentage of 3.8% (A,B) . Convertase activity in absence of properdin was assessed by adding 6.25 μg/ml Salp20 to these samples (C,D) . For P8, P10, P11, and P13, convertase activity was also assessed after compensation for C3 by adding 250 and 500 μg/ml purified C3 to the samples (E–H) . All concentrations indicated have been corrected for the used serum percentage of 3.8%. Representative data of at least two independent experiments are shown. hi-NHS, heat-inactivated NHS.

Article Snippet: A DNA fragment encoding Ixodes scapularis Salp20 [UniProtKB: Q95WZ1 (residue 22–183)] was amplified by PCR from Salp20 synthetic DNA optimized for mammalian expression (GeneART ThermoFisher), and ligated into BamHI-NotI sites of vector pUPE106.03 (U-Protein Express BV, Utrecht, the Netherlands).

Techniques: Activity Assay, Purification

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 1. Leiomodin 1 mRNA and protein expression analysis. A, RT-PCR analysis of Lmod1, Lmod2, and Lmod3 in the indicated adult mouse tissues. Note the similar mRNA expression pattern between Lmod1 and Cnn1, a well known SMC-restricted gene (43). B, quantitative RT-PCR of Lmod1 mRNA in the same tissues as A. Expression levels are displayed as normalized fold increases over brain (set to 1). C, Western blot of LMOD1 protein expression in mouse tissues and in 3T3 cells transfected either with an empty control plasmid or an expression plasmid carrying Lmod1 (top panel). LMOD1 protein expression following preabsorption of the LMOD1 antibody with LMOD1 antigen (middle panel). D, endogenous LMOD1 protein expression in PAC1 SMCs following siRNA knock- downofLMOD1.E,immunohistochemistryofLMOD1proteininaorta(panelsaande),bladder(panelsbandf),brain(panelscandg),andskeletalmuscle(panels d and h) using either the LMOD1 antibody (panels a–d) or a nonimmune, isotype-matched IgG control (panels e–h). The bar in panel h is 30 m for all panels. The data shown in all of the panels are representative of three independent experiments. Arrows in panels c and d indicate LMOD1 positive blood vessels.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 2. LMOD1 protein expression in developing mouse embryos. Immunohistochemistry of LMOD1 protein expression (red stain) in sagittal sections of E9.5 (A), E13.5 (C and D), and E15.5 (E and F) mouse embryos. An isotype-matched IgG control antibody shows no staining of an E9.5 embryo (B). Similar lack of staining was seen with the IgG control applied to E13.5 and E15.5 embryo sections (not shown). ao, aorta; br, bronchiole; bv, blood vessel; he, heart; in, intestine; li, liver; lu, lung; st, stomach. The bars in A and B repre- sent 100 m, and the bars in C–F represent 500 m.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Immunohistochemistry, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 3. Functional analysis of human LMOD1 and LMOD2 promoters. A, VISTA plot indicating nucleotide sequence homology between human (Hsa), mouse (Mmu) and rat (Rno) LMOD1 over a 50-kb genomic interval. The x axis represents the human base sequence, and the y axis indicates the percentage of homology between Hsa versus Mmu (top plot) and Hsa versus Rno (bottom plot). The bent arrow at the top represents the inferred transcription start site. Light teal peaks represent untranslated regions; dark blue peaks represent protein-coding exons; and pink peaks are the conserved nonprotein coding sequences. Sequence logos of two CArG-like elements near LMOD1 are shown below the VISTA plots and illustrate the conservation of each CArG box across six vertebrate species. B and C, LMOD1 and LMOD2 promoter activity in PAC1 SMCs transfected either with SRF-VP16 (B) or MYOCD_v3 (C). The promoter activity reported here and below is a ratio between luciferase and Renilla normalized to that of the pGL3 basic plasmid set to 1. Similar SRF/MYOCD-mediated promoter activity was observed in at least three independent studies.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Functional Assay, Sequencing, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 4. SRF binding to LMOD1 CArG elements. 32P-Labeled double- stranded oligonucleotides containing CArG elements from CNN1 and LMOD1 promoter CArG Far and CArG Near were incubated with the in vitro translated (IVT) SRF in the presence of SRF or MEF2A antibody or a 100-fold molar excess ofunlabeledwildtype(WT)ormutant(Mut)oligonucleotide.SRF-CArGnucle- oprotein complex (SRF) and supershift (SS) are indicated with arrows. Expo- sure times for each EMSA were 11 h (for CNN1) or 64 h (for LMOD1).

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Binding Assay, Labeling, Incubation, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 5. Functional analysis of LMOD1 CArG elements in vitro. A, sche- maticofthewildtype(WT)LMOD1promoterandvariousCArGmutants.Band C, each indicated LMOD1 promoter construct was transfected into PAC-1 SMCs in the presence of SRF-VP16 (B) or MYOCD_v3 (C), and luciferase activity was measured. D, COS-7 cells were similarly transfected with WT or double CArG mutant LMOD1 promoter and either MYOCD_v3, MRTF-A, or MRTF-B. The results shown were reproduced in one independent experiment. *, p 0.001; **, p 0.01.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Functional Assay, In Vitro, Construct, Transfection, Luciferase, Activity Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 6. LMOD1 expression in SRF-deficient cells and tissues. RT-PCR analysis of Lmod1 and Srf mRNA either in PAC-1 SMC transduced with shEGFP or shSRF for 72 h (A) or indicated tissues from adult mice carrying homofloxed Srf alleles and the tamoxifen (Tmx)-inducible Myh11-Cre driver (34) (B). Note the reduction in both Srf and Lmod1 mRNA in tissues derived from mice treated with Tmx (to activate Cre recombinase and effect excision of the Srf gene) versus the vehicle control (Oil). The latter results were extended by comparing protein expression of SRF (C, panels a and b) versus LMOD1 (C, panels c and d) in similar mice treated either with Tmx (C, panels b and d) or sunflower oil (C, panels a and c). The bar in panel d of C is 30 m for all panels. The images in A and B were inverted in Adobe Photoshop so as to better indicate the bands in each gel.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transduction, Derivative Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 8. LMOD1 promoter activity in transgenic mice. Sagittal E12.5 day mouse embryos stained either with -galactosidase to assess LMOD1 promoter activity (A–C and E–G) or an antibody to LMOD1 (D) or control IgG (H). Shown are three WT LMOD1 promoter mice (A–C) and three double CArG mutant LMOD1 promoter mice (E–G). The thick arrows indicate the heart, and the thinner arrows point to aorta and vessels of head. The bar in H is 1 mm for all panels. Eight of 25 wild type founders displayed promoter activity in cardiac muscle, and five of 25 showed promoter activity in vascular tissue. In contrast none of the 10 CArG mutant founders showed LMOD1 promoter activity in cardiac muscle or vascular tissue.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Activity Assay, Transgenic Assay, Staining, Control, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 7. LMOD1 mRNA and protein expression in human cells overexpressing MYOCD. A, the indicated human cell lines were transduced with equal amounts of adenovirus carrying MYOCD or LacZ and endogenous LMOD1 and LMOD2 mRNA expression assessed by RT-PCR. B, same experiment as in A only LMOD1 and MYOCD protein expression were determined using Western blotting. This result was reproduced in an independent experiment.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Transduction, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 3. Functional analysis of human LMOD1 and LMOD2 promoters. A, VISTA plot indicating nucleotide sequence homology between human (Hsa), mouse (Mmu) and rat (Rno) LMOD1 over a 50-kb genomic interval. The x axis represents the human base sequence, and the y axis indicates the percentage of homology between Hsa versus Mmu (top plot) and Hsa versus Rno (bottom plot). The bent arrow at the top represents the inferred transcription start site. Light teal peaks represent untranslated regions; dark blue peaks represent protein-coding exons; and pink peaks are the conserved nonprotein coding sequences. Sequence logos of two CArG-like elements near LMOD1 are shown below the VISTA plots and illustrate the conservation of each CArG box across six vertebrate species. B and C, LMOD1 and LMOD2 promoter activity in PAC1 SMCs transfected either with SRF-VP16 (B) or MYOCD_v3 (C). The promoter activity reported here and below is a ratio between luciferase and Renilla normalized to that of the pGL3 basic plasmid set to 1. Similar SRF/MYOCD-mediated promoter activity was observed in at least three independent studies.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Functional Assay, Sequencing, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 4. SRF binding to LMOD1 CArG elements. 32P-Labeled double- stranded oligonucleotides containing CArG elements from CNN1 and LMOD1 promoter CArG Far and CArG Near were incubated with the in vitro translated (IVT) SRF in the presence of SRF or MEF2A antibody or a 100-fold molar excess ofunlabeledwildtype(WT)ormutant(Mut)oligonucleotide.SRF-CArGnucle- oprotein complex (SRF) and supershift (SS) are indicated with arrows. Expo- sure times for each EMSA were 11 h (for CNN1) or 64 h (for LMOD1).

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Binding Assay, Labeling, Incubation, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 5. Functional analysis of LMOD1 CArG elements in vitro. A, sche- maticofthewildtype(WT)LMOD1promoterandvariousCArGmutants.Band C, each indicated LMOD1 promoter construct was transfected into PAC-1 SMCs in the presence of SRF-VP16 (B) or MYOCD_v3 (C), and luciferase activity was measured. D, COS-7 cells were similarly transfected with WT or double CArG mutant LMOD1 promoter and either MYOCD_v3, MRTF-A, or MRTF-B. The results shown were reproduced in one independent experiment. *, p 0.001; **, p 0.01.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Functional Assay, In Vitro, Construct, Transfection, Luciferase, Activity Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Leiomodin 1, a New Serum Response Factor-dependent Target Gene Expressed Preferentially in Differentiated Smooth Muscle Cells

doi: 10.1074/jbc.m111.302224

Figure Lengend Snippet: FIGURE 6. LMOD1 expression in SRF-deficient cells and tissues. RT-PCR analysis of Lmod1 and Srf mRNA either in PAC-1 SMC transduced with shEGFP or shSRF for 72 h (A) or indicated tissues from adult mice carrying homofloxed Srf alleles and the tamoxifen (Tmx)-inducible Myh11-Cre driver (34) (B). Note the reduction in both Srf and Lmod1 mRNA in tissues derived from mice treated with Tmx (to activate Cre recombinase and effect excision of the Srf gene) versus the vehicle control (Oil). The latter results were extended by comparing protein expression of SRF (C, panels a and b) versus LMOD1 (C, panels c and d) in similar mice treated either with Tmx (C, panels b and d) or sunflower oil (C, panels a and c). The bar in panel d of C is 30 m for all panels. The images in A and B were inverted in Adobe Photoshop so as to better indicate the bands in each gel.

Article Snippet: Prior to immunohistochemistry, the tissues were incubated in antigen retrieval buffer (DAKO) and subjected to steam in a pressure cooker for 10 min. Tissue sections were next blocked in serum-free blocking buffer (DAKO) and then incubated overnight at 4 °C with LMOD1 antibody (Proteintech, 1:1000), SRF (Santa Cruz, 1:1000), or rabbit IgG (Dako, 1:1000).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transduction, Derivative Assay, Control