Samples for western blot were prepared, runned and immunostained as described in [55 (link)]. Human L507 array (Raybiotech, Norcross, GA) was used to detect relative levels of 507 proteins in CMCAAT. Human phospho-kinase antibody array (R&D systems) was used to detect relative phosphorylation levels of 44 kinases. Concentrations of human leptin, adiponectin, IL-6, C-C motif chemokine 22 (CCL22) and colony stimulating factor 1 (CSF-1) in CMCAAT were measured with a Duoset or Quantikine ELISA Kit (R&D systems). Scanning densitometry was carried out with the Quantity One Program (Bio-Rad, Hercules, CA). Functional analysis of detected proteins was performed using FunRich version 2.1 [56 (link)]. Venn diagrams were created with BioVenn software (www.cmbi.ru.nl/cdd/biovenn/ ).
Quantity one program
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Quantity One is a software program designed for imaging and analyzing electrophoresis gels and blots. It provides tools for capturing, processing, and quantifying data from various gel and blot imaging systems.
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Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (5 mentions)
E box cx5 gel documentation system, by Vilber (4 mentions)
Trans blot apparatus, by Bio-Rad (4 mentions)
Molecular imager fx, by Bio-Rad (4 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (4 mentions)
5 bromo 4 chloro 3 indoyl phosphate, by Avantor (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Immobilon fl membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (2 mentions)
Nitro blue tetrazolium, by Avantor (2 mentions)
Polyvinylidene difluoride membrane, by Cytiva (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
Gs 700 imaging densitometer scanner, by Bio-Rad (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
110 protocols using quantity one program
1
Profiling CAAT-conditioned Media Proteins
2
Gelatinase Activity Visualization by Zymography
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MMP-9 gelatinase activity was visualised by zymography. Briefly, SDS polyacrylamide gels (8 %) containing 0.1 % gelatine were overlaid with 4 % stacking gels. Samples of culture media were mixed (10:1 volume) with a sample buffer consisting of 50 mM Tris-HCl, pH 6.8, 2 % SDS, 20 % glycerol, 0.03 % bromophenol blue. After loading the samples, electrophoresis was carried out at 125 V for 2 h. After electrophoresis, the gels were soaked in 2.5 % Triton X-100 on a shaker for 1 h, at room temperature, changing the solution after 30 min, to eliminate SDS. The gels were then equilibrated for 30 min with the digestion buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl 2 , 200 mM NaCl) at room temperature with gentle agitation, then replaced with fresh digestion buffer and incubated at 37 °C overnight. The gels were then stained for 1-2 h with 0.5 % Coomassie Brilliant Blue in 30 % methanol and 10 % acetic acid and destained with 30 % methanol and 10 % acetic acid, after which clear bands of digested gelatine were clearly visible. The gels were scanned by Chemi-Doc (Bio-Rad), utilizing the Quantity One program (Bio-Rad). The densitometric values were normalized with basis on cell proliferation.
3
Fractionation and Analysis of NADPH Oxidase
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Highly aggressively proliferating immortalized (HAPI) cells were stimulated using 0.1 % BSA, 100 nM PMA, or 1 μM each α-Syn peptide and then lysed in a hypotonic lysis buffer (1 mM Tris, 1 mM KCl, 1 mM EGTA, 1 mM EDTA, and 0.1 mM DTT) supplemented with 10 mg/ml of the protease inhibitor mixture (Thermo Scientific, Waltham, MA) and 1 mM PMSF (Thermo Scientific, Waltham, MA). After dounce homogenization (20–25 St, tight pestle A), the cell lysates were centrifuged at 1600×g for 15 min, and the supernatants were further centrifuged at 40,000×g for 2 h. The high-speed centrifugation supernatants, which contain the cytosolic fraction, were then harvested. Meanwhile, the plasma membrane fragments were collected by dissolving the pellets in 1 % nonidet P-40 hypotonic lysis buffer. Proteins in both the high-speed supernatants and the re-suspended pellets were separated by running SDS/PAGE gels and were transferred to polyvinylidene difluoride (PVDF) membranes, followed by immune-blotting using anti-p47phox, anti-p67phox, and anti-gp91phox Abs (Cell Signaling, Danvers, MA). The relative amount of each protein was quantified using the Quantity One program (Bio-Rad, Redmond, WA). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in whole-cell lysates were immunoblotted by an Ab (Cell Signaling, Danvers, MA) as a loading control [21 (link)].
4
Western Blot Analysis of Protein Expression
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Protein was extracted from cells or tissues using lysis buffer containing protease inhibitors. Samples were centrifuged at 12,000×g at 4 °C. Protein concentrations were determined using a BCA Kit (Pierce, Rockford, IL, USA). Protein samples (40 μg) were separated by SDS-PAGE, then transferred to polyvinylidene difluoride membranes. After blocking in 5% fat-free milk, membranes were incubated overnight at 4 °C with the following primary antibodies: anti-MMP-3, anti-SM-MHC, anti-SM-α-actin, anti-MMP-2, anti-SM22 and anti-GAPDH. The antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and used at dilution of 1:1000. Membranes were incubated for 1 h at RT with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Samples were visualized using an Enhanced Chemiluminescence Detection Kit (Amersham Biosciences, Piscataway, NJ, USA). Protein bands were normalized against loading controls, and quantified using the Quantity One program (Bio-Rad, Hercules, CA, USA).
5
Western Blot Analysis of BRCA1, TGF-β1, and Smad3
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Protein from cells or tissues was extracted standardization with a BCA kit (Pierce, Rockford, IL, USA). Then, protein samples (40 μg) were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were then incubated with the following primary antibodies after blocking in 5% nonfat milk: anti-BRCA1 (1:600), anti-p-Smad3 (1:800), anti-Smad3 (1:800), anti-TGF-β1 (1:1000) and anti-β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were then incubated with secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature before the chemiluminescence was measured. The Quantity One program (Bio-Rad, Hercules, CA, USA) was used to measure the intensity of the protein bands.
6
Allelic Loss Analysis of Bap1 in LCM-Isolated Tumor Cells
7
In vitro Transcription Assay Protocol
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Transcription studies of prepared DNA templates were performed with RNA polymerase (RNAP) from Escherichia coli (EcoRNAP) – a holoenzyme complexed RNAP with σ70. The resulted transcripts (RNA) were about 145 nucleobases long. Multiple round in vitro transcription assays were performed essentially as described.29 ,30 (link) The experiments were carried out in total volume 10 μL with 5 ng of DNA template. The reactions proceeded for 10 min at 37 °C after previous preheating of reaction mixture without NTPs. For visualization of prepared RNA product, the transcription was performed in the presence of [α-32P] UTP. The reactions were stopped by the addition of 10 μL of formamide stop solution. The products of transcription were checked by running of 7% polyacrylamide gels. After scanning of exposed gels, the scanned gels were analysed with Quantity One program (BIORAD). For a quantification of relative transcriptions, the transcript signals were normalized to DNA template signals. Signals of transcriptions of modified DNA templates were normalized to the signal of natural DNA (T+ or C+), which was set as 100%. Two–three independent experiments were performed (ESI Section 4.† ).
8
Western Blot Analysis of Wogonin Effects
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Cells were seeded in six-well plates with density of 4 × 105 cells/well, and treated with a vehicle (0.01% DMSO) or 5 and 10 μM wogonin for 72 h. Cell lysates for protein blot analysis were prepared using standard RIPA buffer (Santa Cruz Biotechnology). Western blot analysis was performed to detect the expression levels of target proteins according to the literature (Martínez-Revelles et al., 2016 (link)). Briefly, 30 μg protein was loaded on (10%) SDS-PAGE and separated. After transmembrane, PVDF membranes were stained with primary and secondary antibodies. GAPDH or Histone H3 served as a loading control. The relative intensities of the protein bands were scanned and quantified using Quantity One Program (Bio-Rad).
9
SCD1 Protein Expression Quantification
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For the detection of SCD1, the total protein from the 3T3-L1 cells was extracted using a protein extraction buffer (50 mM NaF, 50 mM Tris HCl, 5 mM NaPPi, 150 mM NaCl, 1% NP-40, 1 mM Na3VO4, and 1 mM EDTA). For each sample, 40 μg of protein was denatured in Laemmli Buffer for 5 minutes at 95°C. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking with a blocking buffer (5% skim milk in PBS), the membranes were incubated with the SCD1 antibody (Cell Signaling Technology, Inc., Danvers, MA, USA). The proteins were visualized using enhanced chemiluminescence with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G. Blots were scanned and analyzed using a multiple image analyzer and the Quantity One program (Bio-Rad Laboratories).
10
AMPK Levels Evaluation in AD Mouse Brains
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AMPK total and phosphorylated AMPK levels in nonTg and 3xTg-AD mice brains were evaluated by western blots analysis [31] . Western blots were performed in the mitochondria-free cytosolic fractions obtained as described above and supplemented with phosphatase inhibitors cocktail (Roche). The samples were resolved by electrophoresis in 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The blots were subsequently incubated with specific primary antibodies [p-AMPK alpha (T172) Antibody from Cell Signaling catalog #2451 (1:1.000); AMPK alpha Antibody from Cell Signaling catalog #2532] overnight at 4 • C. After quick washes with TBS containing 0.1% Tween (TBS-T), the membranes were incubated with secondary antibodies for 2 h at room temperature and again washed with TBS-T. Specific bands of immunoreactive proteins were visualized in a VersaDoc Imaging System (Bio-Rad) after 5-min incubation with enhanced chemifluorescence. The density of protein bands was calculated using the Quantity One Program (Bio-Rad). Results were expressed as the ratio of phosphorylated AMPK to AMPK total.
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