Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: PRIMERS USED TO GENERATE SMALL INTERFERING RNA FROM HUMAN AIRWAY SMOOTH MUSCLE CELL cDNA

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Small Interfering RNA

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: Analysis of the effect of siRNA-induced suppression of integrin α7 mRNA and protein over 6 days of serum-free culture. (A) PCR analysis; α7 PCR product size = 599 base pairs (bp). (B) Western blotting for integrin α7B. Also shown is Western blot analysis of the effects of α7 siRNA on desmin and smooth muscle α-actin accumulation after 6 days of serum deprivation. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; †P < 0.05, compared with control cultures (6-day serum-free media exposed to transfection reagent without siRNA).

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Western Blot, Control, Transfection

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: Western blot analysis showing the effect of siRNA silencing of integrin α7 on the accumulation of integrins α3A (A), α6A (B), α5 (C), and β1 (D) over 6 days of serum deprivation. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; †P < 0.05; ns = not significant compared with control cultures (6-day serum free media exposed to transfection reagent without siRNA). Panels E–H show phase contrast images of human ASM cells in the absence (transfection reagent alone; E) or presence of siRNA directed at integrin α3 (F), α6 (G), or α7 (H). Bar = 50 μm.

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Western Blot, Control, Transfection

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: Western blot analysis showing confirmation of effect of siRNA for integrin α3 (A) or α6 (B) on protein abundance of integrin α3A and α6A. Grouped data, which are expressed as relative units to Day 0, represent results from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; †P < 0.05; ns = not significant compared with control cultures (6 days of serum-free media exposed to transfection reagent without siRNA).

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Western Blot, Quantitative Proteomics, Control, Transfection

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: EFFECTS OF TREATMENT WITH α3 OR α6 INTEGRIN siRNA ON ABUNDANCE OF α5, AND α7B PROTEIN IN 6-DAY SERUM DEPRIVED HUMAN AIRWAY SMOOTH MUSCLE CELL CULTURES

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Quantitative Proteomics

Journal:

Article Title: Laminin-Binding Integrin α7 Is Required for Contractile Phenotype Expression by Human Airway Myocytes

doi: 10.1165/rcmb.2007-0165OC

Figure Lengend Snippet: Western blot analysis showing the effect of siRNA silencing of integrin α3 (A) or integrin α6 (B) on the accumulation of desmin and smooth muscle α-actin over 6 days of serum free culture. Grouped data, which are expressed as relative units to Day 0, represent results obtained from at least four different cultures. *P < 0.05, compared with Day 0, relative to β-actin; ns = not significant compared with control cultures (6 days of serum-free media exposed to transfection reagent without siRNA).

Article Snippet: Thereafter fluorescence micrographs were obtained as we have described ( 3 ) using an Olympus LX70 microscope equipped with charge coupled camera controlled by UltraView Software (Olympus, Hicksville, NY). siRNA Preparation and Study Design The small interfering RNA (siRNA) Generation Kit (Gene Therapy Systems, San Diego, CA) was used to prepare siRNA from human ASM cDNA with primers that amplified integrin α3 cDNA (558 base pairs [bp]), α6 cDNA (523 bp) and α7 cDNA (547 bp, ).

Techniques: Western Blot, Control, Transfection

Journal: European journal of biochemistry

Article Title: Transcriptional regulation of the tissue-type plasminogen-activator gene in human endothelial cells: identification of nuclear factors that recognise functional elements in the tissue-type plasminogen-activator gene promoter.

doi: 10.1046/j.1432-1327.1998.2580123.x

Figure Lengend Snippet: Fig. 5. The predominant tPACRE-binding proteins in C11STH cells are ATF-2, CREB, CREM, c-jun and jun-D. A supershift experiment was performed to identify the tPACRE-binding proteins in C11STH cells. Nuclear extracts prepared from control or PMA-treated C11STH cells were incubated with control buffer (no ab, no antibody) or antibod- ies specific to either ATF-1, ATF-2, CREB, CREM, c-fos, c-jun and jun-D, as indicated (1). Migration profiles were compared on native 5% acrylamide gels as described in Materials and Methods. The four distinct protein/DNA complexes produced in the absence of antibody (C1, C2, C3 and C4) are indicated to the right of the figure by arrows. Antibodies directed against ATF-2, CREB, CREM and c-jun generated supershifted complexes and caused noticeable displacement of complexes from lower positions. PMA treatment caused an increase in the intensity of com- plexes C1 and C4 concomitant with an increase in jun-D binding activity.

Article Snippet: Commercial monoclonal or polyclonal antibodies against ATF-1, ATF-2, CREB, CREM, c-fos, c-jun, Sp-1 and jun-D, specifically designed for supershift experiments, were obtained from Santa Cruz Inc. Supershifting was performed following the same procedure as described for standard band shifting, except that 1 μl specific antibody (1 μg total) was added to the nuclear extracts for 1 h after the addition of the labelled oligomer.

Techniques: Binding Assay, Control, Incubation, Migration, Produced, Generated, Activity Assay

Journal: European journal of biochemistry

Article Title: Transcriptional regulation of the tissue-type plasminogen-activator gene in human endothelial cells: identification of nuclear factors that recognise functional elements in the tissue-type plasminogen-activator gene promoter.

doi: 10.1046/j.1432-1327.1998.2580123.x

Figure Lengend Snippet: Fig. 6. Identification of tPACRE-binding proteins in HUVECs. Nuclear extracts prepared from non-treated and PMA-treated HUVECs were used in a supershift experiment using a labelled tPACRE as a probe. Antibodies specific for either ATF-1, ATF-2, CREB, CREM, c-fos, c-jun and jun-D were included as indicated (1) and the migration profiles were compared on native 5% acrylamide gels. The position of complexes C1, C2, C3 and C4 are indicated to the left of the figure. Antibodies directed against ATF-2, CREB, CREM and c-jun generated supershifted complexes and caused noticeable displacement of com- plexes from lower positions in a manner similar to that seen in Fig. 5 using nuclear proteins extracted from C11STH cells. The effect of PMA was less striking and caused a slight increase in the tPACRE-binding activity of jun-D.

Article Snippet: Commercial monoclonal or polyclonal antibodies against ATF-1, ATF-2, CREB, CREM, c-fos, c-jun, Sp-1 and jun-D, specifically designed for supershift experiments, were obtained from Santa Cruz Inc. Supershifting was performed following the same procedure as described for standard band shifting, except that 1 μl specific antibody (1 μg total) was added to the nuclear extracts for 1 h after the addition of the labelled oligomer.

Techniques: Binding Assay, Migration, Generated, Activity Assay

Journal: European journal of biochemistry

Article Title: Transcriptional regulation of the tissue-type plasminogen-activator gene in human endothelial cells: identification of nuclear factors that recognise functional elements in the tissue-type plasminogen-activator gene promoter.

doi: 10.1046/j.1432-1327.1998.2580123.x

Figure Lengend Snippet: Fig. 7. Identification of endothelial nuclear factors that recognise the Sp-1 site in the t-PA promoter. Nuclear extracts prepared from C11STH cells and HeLa cells were used in a supershift experiment using a labelled oligomer corresponding to the region between position 160 and position 174 in the t-PA promoter as a probe which includes the Sp-1-binding site. Antibodies specific for Sp-1 were included as indi- cated (1). The arrow to the left of the figure indicates the position of the major protein/DNA complex supershifted by the anti-Sp-1 antibody.

Article Snippet: Commercial monoclonal or polyclonal antibodies against ATF-1, ATF-2, CREB, CREM, c-fos, c-jun, Sp-1 and jun-D, specifically designed for supershift experiments, were obtained from Santa Cruz Inc. Supershifting was performed following the same procedure as described for standard band shifting, except that 1 μl specific antibody (1 μg total) was added to the nuclear extracts for 1 h after the addition of the labelled oligomer.

Techniques: Binding Assay

Journal: Genome Biology

Article Title: Assessment of computational methods for the analysis of single-cell ATAC-seq data

doi: 10.1186/s13059-019-1854-5

Figure Lengend Snippet: Schematic overview of single-cell ATAC-seq assays and analysis steps. a Single-cell ATAC libraries are created from single cells that have been exposed to the Tn5 transposase using one of the following three protocols: (1) Single cells are individually barcoded by a split-and-pool approach where unique barcodes added at each step can be used to identify reads originating from each cell, (2) microfluidic droplet-based technologies provided by 10X Genomics and BioRad are used to extract and label DNA from each cell, or (3) each single cell is deposited into a multi-well plate or array from ICELL8 or Fluidigm C1 for library preparation. b After sequencing, the raw reads obtained in .fastq format for each single cell are mapped to a reference genome, producing aligned reads in .bam format. Finally, peak calling and read counting return the genomic position and the read count files in. bed and .txt format, respectively. Data in these file formats is then used for downstream analysis. c ATAC-seq peaks in bulk samples can generally be recapitulated in aggregated single-cell samples, but not every single cell has a fragment at every peak. A feature matrix can be constructed from single cells (e.g., by counting the number of reads at each peak for every cell). d Following the construction of the feature matrix, common downstream analyses including visualization, clustering, trajectory inference, determination of differential accessibility, and the prediction of cis -regulatory networks can be performed using the methods benchmarked in this manuscript

Article Snippet: As many of our evaluated methods were designed in the context of data generated from the Fluidigm C1 platform (which produces ~ 10 2 cells), such approaches were often incapable of analyzing large datasets.

Techniques: Sequencing, Construct