GLAP was synthesized according to a slightly modified method of Usui et al. [28 (link)]. In brief, glyceraldehyde (0.2 M) and N-acetyl-L-lysine (0.1 M) were dissolved in 0.2 M sodium phosphate buffer (pH 7.4), and incubated at 37°C for a week. The reaction mixture was filtered with a polyvinylidene difluoride membrane filter (0.22 mm, Millipore, Bedford, MA, USA), and then put on a C8 column on preparative reversed phase high-performance liquid chromatography (HPLC). HPLC was done with a quaternary gradient pump PU-2089 plus (JASCO Co. Ltd., Tokyo, Japan) and monitoring at 215 nm with the UV–VIS spectrophotometric detector UV-2075 plus (JASCO Co., Ltd. Tokyo, Japan) under the following conditions: Column: COSMOSIL 5C8-AR-300 column (250 × 20 mm I.D., Nacalai Tesque Inc., Kyoto, Japan). Elution: isocratic of 25 mM sodium phosphate buffer (pH 7.0) from 0 to 30 min and a linear gradient of 0-40% acetonitrile containing 25 mM sodium phosphate buffer (pH 7.0) from 30 to 60 min. Flow rate: 2 ml/min. Temperature: ambient.
Polyvinylidene difluoride filter membrane
Manufactured by Merck Group
Sourced in United States
Polyvinylidene difluoride (PVDF) filter membranes are a type of lab equipment used for filtration applications. They are made of a chemically resistant and durable polymer material that is suitable for a variety of filtration processes. PVDF membranes have a porous structure that allows for efficient separation and retention of targeted analytes or particles from liquid samples.
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Lab products found in correlation
Typhoon 8600 imager, by GE Healthcare (4 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Snail, by Abcam (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Vimentin, by Abcam (2 mentions)
N cadherin, by Abcam (2 mentions)
Alkaline phosphatase conjugated goat anti mouse igg antibody, by Santa Cruz Biotechnology (2 mentions)
Enhanced chemiluminescence system, by Merck Group (2 mentions)
Uv 2075 plus, by Jasco (1 mentions)
Pu 2089 plus, by Jasco (1 mentions)
Cyclin d1, by Merck Group (1 mentions)
Image lab 3, by Bio-Rad (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
P21cip1, by Santa Cruz Biotechnology (1 mentions)
Phospho p90rsk, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
19 protocols using polyvinylidene difluoride filter membrane
1
Synthesis and Purification of GLAP
2
Protein Analysis of MAPK Pathway
3
Western Blot Analysis of Tendon Proteins
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The tendon samples were homogenized. Protein content was normalized and the samples were subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane filter (Millipore Corp., Billerica, Mass.). The filters were incubated in phosphate-buffered saline containing 0.5% Tween 20 and 5% nonfat milk and then incubated with primary antibody overnight at 4 °C. After incubation with conjugated affinity-purified secondary antibody labeled with IRDye 800, blots were washed and immunoreactive proteins were scanned on an Odyssey imager (LI-COR, Inc., Lincoln, NE). Optical density on the membrane was measured and the relative differences between an internal control (ß-actin) and treated samples were calculated. Mouse anti-rat bFGF (Milipore Corp., Billerica, Mass.), mouse anti-human VEGF (Santa Cruz, Dallas, Texas), mouse anti-chicken MMP2 and TIMP2 (Abcam, Cambridge, Mass.) and mouse anti-chicken type I collagen and type III collagen (Acris, San Diego, Calif.) were used respectively as primary antibodies to detect different proteins.
4
Immunoprecipitation and Western Blotting
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Cells were collected and lysed in lysis buffer containing 250 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.5% NP-40, 2 mM Na3VO4, 10 mg ml−1 leupeptin, 10 mg ml−1 trypsin inhibitor, 10 mg ml−1 aprotinin and 2 mM phenylmethylsulfonyl fluoride. For immunoprecipitation, cell lysates were incubated with respective antibodies and protein G-beads (GE Healthcare). The beads were then washed six times with the lysis buffer, and the immune complex was eluted with SDS–polyacrylamide gel electrophoresis (PAGE) sample buffer. The lysates and immunoprecipitates were subjected to SDS–PAGE followed by immunoblotting. Proteins were transferred to a polyvinylidene difluoride membrane filter (Millipore) and visualized using western blot chemiluminescence reagent (Perkin-Elmer Life Sciences). The obtained chemiluminescence was exposed to X-ray film (GE Healthcare). Uncropped images for immunoblots are shown in Supplementary Fig. 8 .
5
Detecting MCM Proteins in Glioma Tumors
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To detect MCM2, MCM3 and MCM7 protein in glioma tumors, we examined six glioma samples (two each of WHO stages II, III, IV). The protein concentration of cell lysates was determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Thirty micrograms of protein per tissue lysate was electrophoresed on 7-12% gels and transferred to a polyvinylidene difluoride membrane filter (Millipore, Billerica, MA, USA). Blots were probed with the appropriate antibody. Quantitative determination of the protein was assessed by densitometric scanning of the band from film. An AlphaImager 2000 Documentation and Analysis System (Alpha Innotech Corp, San Leandro, CA, USA) was used to quantify bands of appropriate sizes.
The following primary antibodies were used: anti-β-actin (Sigma Chemical Co, St. Louis MO, USA), anti-MCM2 (sc-10771, Santa Cruz Biotechnology, USA), anti-MCM3 (sc-9849, Santa Cruz Biotechnology, USA), and anti-MCM7 (sc-22782, Santa Cruz Biotechnology, USA).
The following primary antibodies were used: anti-β-actin (Sigma Chemical Co, St. Louis MO, USA), anti-MCM2 (sc-10771, Santa Cruz Biotechnology, USA), anti-MCM3 (sc-9849, Santa Cruz Biotechnology, USA), and anti-MCM7 (sc-22782, Santa Cruz Biotechnology, USA).
6
Serum-free culture of NSCLC cell lines
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Serum-free culture was performed as described previously (8 (link)) with slight modifications. The NSCLC cell lines were trypsinised, spun down at 4°C and washed twice with cold serum-free MCDB-104GK medium (Nihon Pharmaceutical Co., Ltd.). The cell lines were then serum-deprived, seeded at a density of 0.2 or 2×105 cells/cm2 in 35-mm dishes (Asahi Techno Glass), 60-mm dishes (Asahi Techno Glass) or 100-mm dishes (Becton Dickinson Labware) in serum-free MCDB-104GK medium supplemented with penicillin (5 µg/ml), streptomycin (5 µg/ml) and neomycin (10 µg/ml) and incubated for 24 h. The serum-free culture supernatant derived from EBC-1 cells (EBC-1 supernatant) and the cells were spun down, and each supernatant was filtrated through a 0.45-µm polyvinylidene difluoride membrane filter (Merck Millipore, Italy) and concentrated by ultrafiltration (Amicon Ultra 3K, Merck Millipore). Protein concentrations in EBC-1 supernatants were determined using the Bradford method.
7
Characterization of Amla Fruit Extract
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A commercial product of Amla fruit juice extract (SunAmla) was obtained from Taiyo Kagaku Co., Ltd. (Mie, Japan). It is a dried powder of water extract from fresh Amla fruit and was previously shown to contain ~30% polyphenols and 2% vitamin C [6 (link)]. To confirm the equivalence of Amla used in this study, total polyphenol was analyzed by a colorimetric method using gallic acid as a standard [6 (link)], and vitamin C content was analyzed by high-performance liquid chromatography (HPLC) as previously reported [16 (link)]. Glucose and fructose in Amla were analyzed by HPLC as previously reported [17 (link)]. To prepare Amla extract stock solution, the extract was dissolved in distilled water at a concentration of 200 mg/mL and then sterilized using a polyvinylidene difluoride membrane filter (Merck Millipore, Darmstadt, Germany).
8
Geodia barretti Sponge and Seawater Sampling
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Norway Geodia barretti (G. barretti NOR) samples (gb1-gb10) were collected and processed by E. Peters et al. (unpublished data), and Canada Geodia barretti (G. barretti CAN) samples (gb126, gb278, gb305) were collected and processed by Steffen et al. (107 (link)). Atlantic Seawater (Seawater ATL) samples (gb1_f – gb10_f) were collected onboard R/V Hans Brattström of the University of Bergen from Korsfjord, Bergen, Norway (60°8.13′N, 5°6.7′E) in September and October 2017 by filtering 2 L of seawater through polyvinylidene difluoride membrane filters (pore size, 0.22 μm; diameter, 47 mm; Merck Millipore, Burlington, MA). Filters were snap-frozen in liquid nitrogen and stored at −80°C. Aplysina aerophoba and Mediterranean Seawater (Seawater_MED) samples were collected and processed as described by Chaib De Mares et al. (88 (link)) and are publicly available (88 (link)). Petrosia ficiformis sampling took place in August 2018 at a semisubmerged marine cave (5- to 6-m depth) with internal freshwater springs in Sfakia, Greece (35°12′N, 24°7′E) in a collaborative effort with the Hellenic Centre for Marine Research (HCMR). Immediately after collection, the samples were transferred to liquid nitrogen and stored at −80°C.
9
Western Blot Analysis of Protein Markers
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Proteins were extracted from the tissue samples and cell lines, and the total protein concentration was determined using a Pierce bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (30 μg) were fractionated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride membrane filters (EMD Millipore, Billerica, MA, USA). Next, 10% non‐fat dry milk was used to block nonspecific binding. The membranes were incubated with primary antibodies overnight. Following washing, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase anti‐mouse/rabbit immunoglobulin G (Boster Biological Technology Co. Ltd., Pleasanton, CA, USA). Finally, the protein levels were quantified using an enhanced chemiluminescence detection system (Amersham imager 600; General Electric, Fairfield, CT, USA). The antibodies used in this study were as follows: FGFR4 (1:1000; Santa Cruz Biotechnology); N‐cadherin, Vimentin, Snail, ERK, p‐ERK, p‐FGFR4, and Bcl‐2 (1:1000; Abcam, Cambridge, MA, USA); E‐cadherin (1:1000; Affbiotech, Cincinnati, OH, USA); Claudin‐1, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), and Bax (1:1000; Proteintech, Chicago, IL, USA); β‐actin (1:1000; Boster Biological Technology Co. Ltd.); and AKT and p‐AKT (1:1000; Omimabs, Alhambra, CA, USA).
10
Western Blot Analysis of EMT Markers
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Proteins were extracted from the tissue samples and cell lines, and the total protein concentration was determined using a bicinchoninic acid kit. Equal amounts of protein (40 µg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride membrane filters (EMD Millipore, Billerica, MA, USA). Next, 5% non-fat dry milk was used to block nonspecific binding. The membranes were incubated with primary antibodies. Following washing, the membranes were incubated with the corresponding secondary antibodies conjugated with horseradish peroxidase. Finally, the protein levels were quantified using an enhanced chemiluminescence detection system (Amersham imager 600; General Electric, Fairfield, CT, USA). The antibodies used in this study were as follows: CEP55, N-cadherin, Vimentin and Snail (1:1,000; Abcam Inc.); E-cadherin (1:1,000; Affbiotech, Cincinnati, OH, USA); Claudin-1 and GAPDH (1:1,000; Proteintech, Chicago, IL, USA); Akt (1:1,000; Omimabs, Alhambra, CA, USA) and pAktS473 and pAktT308 (1:1,000; Cell Signaling Technology, Danvers, MA, USA).
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