For amplification of porcine OPN promoter methylation analysis site, PCR was performed using 2 μL of bisulfite-converted genomic DNA as template. The primer sets of COBRA were OPN-C sense 5′-TTTTTTGAGGGAGATTAGTTTTTG-3′ and antisense 5′-ATTCTACTAAAATCCAACCACCC-3′. The COBRA-PCR products were purified by phenol/chloroform, followed by ethanol precipitation. The DNA was resuspended in 8.5 μL of distilled deionized water. Purified PCR products were then digested with 10 U BstUI restriction enzyme (New England Biolabs, MA, USA) at 65°C. Products were electrophoresed on 6% native acrylamide gel, stained with 200 g/mL ethidium bromide, and visualized using a Kodak 1D software.
1d software
Manufactured by Kodak
Sourced in United States
Kodak 1D software is a data analysis and visualization tool designed for laboratory use. It provides capabilities for processing and analyzing one-dimensional data, such as spectroscopic or chromatographic measurements. The software offers basic data manipulation and graphing functions to support scientific research and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Merck Group (2 mentions)
Transwell system, by Corning (2 mentions)
Bstui restriction enzyme, by New England Biolabs (1 mentions)
Paxillin, by BD (1 mentions)
Trueblot immunoprecipitation beads, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Developmental Studies Hybridoma Bank (1 mentions)
Rabbit anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Mmp 2, by Merck Group (1 mentions)
Mmp 9, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Immobilon, by Merck Group (1 mentions)
Electrophoresis reagents, by Bio-Rad (1 mentions)
Random primer, by Promega (1 mentions)
Gotaq flexi polymerase, by Promega (1 mentions)
9 protocols using 1d software
1
Porcine OPN Promoter Methylation Analysis
2
Quantitative Analysis of Protein Interactions
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For immunoprecipitation studies, equal amounts of protein were used from ex vivo epithelial explants extracted in TX/OG from different days in culture or between treated and untreated samples. For immunoprecipitations, samples were incubated at 4°C sequentially with primary antibody and TrueBlot Immunoprecipitation beads (eBioscience, San Diego, CA) and the immunoprecipitates subjected to SDS/PAGE as described previously and earlier (Walker et al., 2002 (link)). Antibodies used included vimentin (Developmental Studies Hybridoma Bank) and paxillin (BD Transduction Laboratories). Immunoblots were scanned and densitometric analysis was performed using Kodak 1D software. To standardize these results for the paxillin IP, quantitative analysis was performed. For each protein analyzed, the densitometry results were normalized to D1 OAZ, and then the ratio of each coprecipitated protein to the original immunoprecipitated protein was calculated for each sample. For vimentin IP, the ratio of myosin IIA versus myosin IIB associated with vimentin was calculated for each sample.
3
Quantification of Leptin and NMDAR Receptor Levels
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The total protein concentrations in the punched ARCN and PVN samples were measured with a bicinchoninic acid assay kit (Pierce, Rockford, IL). Samples were adjusted to contain the same concentration of total protein. The protein samples with 2X 4% SDS sample buffer were loaded onto a SDS-PAGE gel, subjected to electrophoresis, and then transferred to a polyvinylidene difluoride membrane (Millipore, MA). The membrane was probed with primary antibody [rabbit anti-leptin receptor (1 : 500, Abcam, Cambridge, MA), rabbit anti-NR1 receptor (1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA), or rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1 : 2000, Santa Cruz Biotechnology)] overnight and then probed with secondary antibody (peroxidase-conjugated anti-rabbit IgG, 1 : 5000, Pierce). An enhanced chemiluminescence substrate (Pierce) was applied to the membrane, followed by an exposure within an UVP system (UVP BioImaging, Upland, CA) for visualization. Kodak 1D software (Kodak, NY) was used to quantify the signal. The expression of protein was calculated as the ratio of intensity of the leptin receptor and NR1 receptor, respectively, relative to the intensity of GAPDH band.
4
Western Blot Analysis of Extracellular Matrix Proteins
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Samples were extracted and lysed in TX/OG buffer (44.4 mM n-octyl β-d -glucopyranoside, 1% Triton X-100, 100 mM NaCl, 1 mM MgCl2, 5 mM EDTA, 10 mM imidazole) containing 1 mM sodium vanadate, 0.2 mM H2O2, and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations were determined with the bicinchoninic acid assay (Pierce, Rockford, IL). Proteins were separated on Tris-glycine gels (Novex, San Diego, CA), electrophoretically transferred to membrane (Immobilon-P; Millipore, Billerica, MA), and immunoblotted as described previously (Walker and Menko, 1999 (link)). For detection, ECL reagent (Amersham Life Sciences, Arlington Heights, IL) was used. Immunoblots were scanned, and densitometry analysis was performed (1D software; Eastman Kodak, Rochester, NY). The ratio of each protein to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was calculated for each sample and normalized to the ratio at day 1 in the CMZ and graphed ±SEM. Gels for CD44 analysis were run under nonreducing conditions; gels for MMP-9 and MMP-2 analysis were run under reducing conditions. Antibodies used for Western blotting included CD44 (monoclonal antibody [mAb]; Developmental Studies Hybridoma Bank, Iowa City, IA), MMP-9 (polyclonal; Millipore), MMP-2 (monoclonal; Millipore), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA).
5
Transwell Assay for Cell Migration
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Cell migration was assayed using the Transwell system (Corning Costar, Acton, MA, USA) with 6.5 mm-diameter polycarbonate filters (8-µm pore size). Briefly, the lower surface of the filter was coated with 10 µg/mL fibronectin (Sigma-Aldrich Corp.), 10 µg/mL recombinant human TGFBIp (rhTGFBIp) (Sino Biological Inc., Beijing, China), or bovine serum albumin at a concentration of 3% (w/v) as a control for nonspecific binding. SV40-CECs or primary CECs (105) were seeded onto chemotaxis filters in DMEM plus 1% fetal bovine serum. After the 5-hour migration period, non-migrating cells were removed and migrating cells were stained with hematoxylin and eosin (H&E) and quantified using Kodak 1D software (Eastman Kodak, Rochester, NY, USA). Results are representative of three different experiments in duplicate.
6
Recombinant C3 Protein Binding Analysis
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4 μg of recombinant C3 protein were separated by native 15% PAGE and transferred to nitrocellulose membranes. After Western blot the transfer efficiency was checked with Ponceau S staining. After incubation with blocking buffer (5% powdered milk, in TBST), membranes were probed with 10 μg/ml of purified His-tagged vimentin in blocking buffer for 1 h at 4 °C. After extensive washing with TBST, membranes were incubated with C3 antibody or α-His, followed by HRP-conjugated secondary antibody, and detected by ECL. For the chemiluminescence reaction, ECL Femto (Pierce, Thermo Fisher Scientific Inc.) or Immobilon (Millipore, Schwalbach, Germany) were used. All signals were analyzed densitometrically using the KODAK 1D software (KODAK GmbH, Stuttgart, Germany).
7
Western Blotting for NSP and PAI-1 Detection
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Both the mini-protean three apparatus used for electrophoresis and protein transfer, as well as electrophoresis reagents were from BioRad (Hercules, CA, USA). Polyacrylamide gels were cast with 3% stacking and 10% separating gels and Western blotting for NSP or PAI-1 was carried out as previously described (Barker-Carlson et al., 2002 (link); Li et al., 2008 (link)). Analysis of immunoblots using known amounts of NSP that was intact, cleaved, or in complex with sct-PA demonstrated no detectable difference in epitope detection among the states of NSP (Barker-Carlson et al., 2002 (link)). As noted in our previous paper (Barker-Carlson et al., 2002 (link)) these reagents were validating as demonstrating no detectable difference in epitope detection among the states of NSP. This information is included in the “Methods” section. A standard curve for NSP-sc-tPA complex detection using this methodology is also included in Supplemental Figures S1A,B . Kodak 1D Software was used to image all films, and Prism 3.0 or 4.0 software were used for data analysis as indicated.
8
Cell Migration Assay Using Transwell
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Cell migration was assayed using the Transwell system (Corning Costar, Acton, MA, USA) using 6.5 mm-diameter polycarbonate filters (8-µm pores). The lower surface of the filter was coated with 10 µg/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA), and LECs (105 cells) from passage 3 were seeded onto chemotaxis filters in EBM supplemented with 0.5% FBS. After 5 h incubation, non-migrating cells were removed and migrating cells were analyzed by hematoxylin and eosin (H&E) staining. Quantification was performed using Kodak 1D software (Eastman Kodak, Rochester, NY, USA). The results were performed in triplicate with three different cell lines.
9
RT-PCR for Transcription Quantification
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RT was performed in a 20-µl reaction mixture containing 2.5 µg of total RNA, random primers at 200 ng/µg of RNA (Promega Corp.), and 30 U of Avian myeloblastosis virus reverse transcriptase, following the manufacturer's instructions. Conditions used for RT were 65°C for 3 min, 25°C for 10 min, 42°C for 90 min, and 70°C for 10 min. The primers 16s_M130_Rv/Fw were used to measure the transcription of 16S rRNA. Second-strand synthesis was performed using GoTaq Flexi polymerase (Promega Corp.) with 1 µl of undiluted (any test gene) or 1:1,000 diluted (16S rRNA) cDNA reaction as template. The number of PCR cycles for each gene was standardized so that the product amplification was in the linear range; 10 to 20 µl of the PCR product was analyzed by agarose gel electrophoresis. The intensities of the bands were measured and normalized to that of 16S rRNA using Kodak 1D software (Pizzonia 2001 ) to obtain the fold difference. The validation of each gene was performed with samples from three independent isolations.
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